Synthetic rig-i-like receptor agonists

ABSTRACT

The present disclosure relates to, inter alia, RNA molecules (e.g., RNA hairpin agonists) that bind to and agonize RIG-I-like receptors (RLRs), and to use of the molecules in methods for treating, or ameliorating one or more symptoms of, a disorder (e.g., cancer).

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/659, 999, filed on Apr. 19, 2018. The entire content of the aforementioned application is incorporated herein by this reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created Nov. 5, 2019, is named “CMN-002_SequenceListing” and is 46937 bytes in size.

BACKGROUND

Exogenous nucleic acids, particularly viral nucleic acids, introduced into cells induce an innate immune response, resulting in, among other events, interferon (IFN) production and cell death. Upon sensing viral RNA, RIG-I-like receptors induce type I interferon (IFN) secretion leading to upregulation of antiviral IFN-induced proteins in the infected and neighboring cells, which inhibits virus replication. Further downstream events attract immune cells and trigger the adaptive immune response. In addition, RIG-I ligands have been reported to induce the apoptosis of many different types of tumor cells, but not of normal cells.

There remains a need for additional and improved compositions and methods to modulate the activity of immunomodulatory proteins. Such agents can be used for cancer immunotherapy and treatment of other conditions, such as chronic infection. There is a need to develop improved RIG-I-like receptor ligands for diverse therapeutic immunomodulatory applications.

SUMMARY OF THE DISCLOSURE

The present disclosure is based, at least in part, on the discovery of synthetic RNA molecules that function as RIG-I-like receptor agonists.

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, and wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif. In some embodiments, the first polynucleotide comprises the sequence motif.

In some embodiments the RLR agonists of the disclosure comprise a sequence motif selected from the group consisting of:

(i) a GT-repeat motif;

(ii) a GA-repeat motif;

(iii) a AUCG-repeat motif;

(iv) an AU-repeat motif;

(v) a dipyrimidine motif;

(vi) a dipurine motif;

(vii) a pyrimidine triplet motif;

(viii) a purine triplet motif;

(ix) a palindromic sequence motif; and

(x) a combination of any of (i)-(ix).

In some embodiments, the RLR agonists of the disclosure comprise a combination of sequence motifs. In some embodiments the combination of sequence motifs is a GT-repeat motif and a purine triplet motif In some embodiments, the combination of sequence motifs is an AUCG-repeat motif and a dipyrimidine motif. In some embodiments, the combination of sequence motifs is an AUGC-repeat motif and a dipurine motif.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the at least one improved biological activity is selected from:

(i) an increase in RLR-mediated cytokine production;

(ii) an increase in RLR-mediated expression of interferon-stimulated genes;

(iii) an increase in RLR-mediated intracellular signaling;

(iv) an increase in binding affinity to RLRs; and

(v) a combination of any of (i)-(iv).

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif that increases RLR-mediated type I interferon (e.g., IFN-α, IFN-(3) production relative to an agonist that does not comprise the sequence motif. In some embodiments, the RLR agonists of the disclosure comprise a sequence motif that increases RLR-mediated IL-1β production relative to an agonist that does not comprise the sequence motif. In some embodiments, the RLR agonists of the disclosure comprise a sequence motif that increases RLR-mediated IP-10 production relative to an agonist that does not comprise the sequence motif In some embodiments, the RLR agonists of the disclosure comprise a sequence motif that increases RLR-mediated IL-6, IL-12p70, MCP-1 and/or MIP-1β production relative to an agonist that does not comprise the sequence motif.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a GT-repeat motif (e.g., GTGTGT) comprising a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of <19 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 15-18 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 15 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 10-15 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 5-10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 5 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of about 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some aspects, the GT-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a GT-repeat motif comprising a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 18 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 16 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 14 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 12 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 10 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 8 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 6 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GT-repeat motif comprising a sequence of 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a GT-repeat motif, wherein the GT-repeat motif is [GT]_(n), wherein n=2 to 9, 3-7, or 4-8. In some aspects, the GT-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some aspects, the disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to (operably linked to) a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a GT-repeat motif comprising a sequence of about 14 guanine and thymine nucleotides. In some embodiments, the sequence motif is a GT-repeat motif, wherein the GT-repeat motif is [GT]₇. In some aspects, the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some aspects, the disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a GT-repeat motif comprising a sequence of 6 guanine and thymine nucleotides. In some embodiments, the sequence motif is a GT-repeat motif, wherein the GT-repeat motif is [GT]₃. In some embodiments, the sequence motif is a GT-repeat motif, wherein the GT-repeat motif is [GT]₃, and wherein the GT-repeat is followed by a purine triplet and UCG, respectively. In some embodiments, the purine triplet is GGA. In some aspects, the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a GA-repeat motif (e.g., GAGAGA) comprising a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of <19 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 15-18 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 15 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 10-15 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 10 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 5-10 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 5 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of about 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some aspects, the GA-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif is a GA-repeat motif comprising a sequence of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 18 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 16 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 14 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 12 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 8 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 6 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a GA-repeat motif comprising a sequence of 4 guanine and adenine nucleotides, or derivatives or analogs thereof.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a GA-repeat motif, wherein the GA-repeat motif is [GA]_(n), where n=2 to 9, 3 to 7 or 4 to 8. In some aspects, the GA-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some aspects, the disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a GA-repeat motif comprising a sequence of about 14 guanine and adenine nucleotides. In some embodiments, the sequence motif is a GA-repeat motif, wherein the GA-repeat motif is [GA]₇. In some aspects, the GA-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a AUCG-repeat motif (e.g., AUCGAUCG) comprising a sequence of <19, about 16, about 12-16, about 12, about 8-12, about 6, 16, 12, 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of <19 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of about 16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of about 12-16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of about 12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of about 8-12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of about 6 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of 16 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of 12 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some embodiments, the sequence motif is a AUCG-repeat motif comprising a sequence of 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof. In some aspects, the AUCG-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]_(n), where n=2 to 4 or 2, 3 or 4. In some aspects, the AUCG-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some aspects, the disclosure provides an RLR agonist that specifically binds to an RLR, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, wherein the first polynucleotide comprises the sequence motif, and wherein the sequence motif is a AUCG-repeat motif comprising a sequence of about 12 guanine and adenine nucleotides. In some embodiments, the AUCG-repeat motif is [AUCG]₃. In some aspects, the AUCG-repeat motif provides an improved biological activity in the RLR agonist, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise a AUCG-repeat motif, wherein the motif is preceded by a CG or a dipyrimidine motif In some embodiments, the AUCG-repeat motif is preceded by a CG. In some embodiments, the AUCG-repeat motif is [AUCG]₃ and is preceded by a CG. In some embodiments, the AUCG-repeat motif is [AUCG]₃ and is preceded by the dipyrimidine motif CC.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein the motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the AUCG-repeat motif is [AUCG]₃ and is preceded by the dipurine motif GA. In some embodiments, the AUCG-repeat motif is preceded by the dipurine motif II.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein one or more uridine nucleosides (U) are substituted with a modified nucleoside. In some embodiments, wherein the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC-repeat motif is [AUCG]₃, wherein the one or more uridine nucleosides (U) comprising the AUCG-repeat motif are substituted with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC-repeat motif is [AUCG]₃, wherein the one or more uridine nucleosides (U) comprising the AUCG-repeat motif are substituted with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T), and wherein the AUGC-repeat motif is preceded by GG.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein one or more guanosine nucleosides (G) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is inosine (I). In some embodiments, the AUGC-repeat motif is [AUCG]₃, wherein the one or more guanosine nucleosides (G) comprising the AUCG-repeat motif are substituted with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T), and wherein the AUGC-repeat motif is preceded by GG.

In some embodiments, the RLR agonists of the disclosure comprise a AUCG-repeat motif, wherein the motif is preceded by a IG. In some embodiments, the AUCG-repeat motif is [AUCG]₃ and is preceded by a IG.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat, wherein one or more guanosine nucleosides (G) are substituted with an inosine (I), wherein the AUCG-repeat is preceded by an inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG-repeat are substituted with an inosine (I), wherein the AUCG-repeat is preceded by an inosine (I), wherein the 5′ most nucleotide of the first polynucleotide comprises inosine (I).

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂. In some embodiments, the sequence motif is an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂, and wherein the AUCG-repeat motif is preceded by a dipurine motif In some embodiments, the sequence motif is an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂, wherein the AUCG-repeat motif is preceded by a dipurine motif, and wherein the dipurine motif is GG.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂, and wherein the AUCG-repeat motif is preceded by a purine triplet motif In some embodiments, the purine triplet motif is GGG. In some embodiments, the sequence motif is an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂, wherein the AUCG-repeat motif is preceded by a purine triplet motif, and wherein the purine triplet motif is GGG. In some embodiments, the sequence motif is an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂, and wherein the AUCG-repeat motif is preceded by CCCCCG. In some embodiments, the sequence motif is an AUCG-repeat motif, wherein the AUCG-repeat motif is [AUCG]₂, and wherein the AUCG-repeat motif is preceded by TCGUCG.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a palindromic sequence comprising a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of <19 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 15-18 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 15 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 10-15 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of about 10 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 18 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence 17 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 16 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 15 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 14 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 13 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 12 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 11 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 10 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 9 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 8 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 7 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 6 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 5 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome. In some embodiments, the sequence motif is a palindromic sequence comprising a sequence of 4 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome.

In some embodiments, the RLR agonists of the disclosure comprise a linker, wherein the linker is flanked by AU. In some embodiments, the linker is flanked by an AU-repeat motif, wherein the AU-repeat motif is [AU]_(n), where n=2 to 3. In some embodiments, the AU-repeat motif is [AU]₂.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

-   -   (N₁-N₂-X₁) comprises a first polynucleotide comprising linked         nucleotides N₁, N₂ and X₁;     -   (ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising         linked nucleotides X₂, N₃ and N₄;     -   (iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide         comprising a nucleoside selected from the group consisting of:         adenosine, guanosine, cytidine, 5-methyluridine, uridine and         inosine;     -   (iv) N₁ base pairs with N₄;     -   (v) N2 base pairs with N₃;     -   (vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or         derivative or analog thereof;     -   (vii) X₁ and X₂ are each oligonucleotides comprising nucleosides         selected from the group consisting of: adenosine, guanosine,         cytidine, 5-methyluridine, uridine and inosine;     -   (viii) X₁ is complementary to X₂;     -   (ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in         length and are the same length, and;     -   (x) L is a linker that operably links the first polynucleotide         and the second polynucleotide,

wherein at least one of N1, N2, N3, and N4 is inosine and/or at least one of X1 and/or X2 comprises at least one inosine nucleoside, and wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA. In some aspects, the RLR agonist of the disclosure has an improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a non-nucleotide linker, and wherein the agonist comprises the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X2-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is the non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

wherein inosine, if present, base pairs with cytidine. In some aspects, the RLR agonist of the disclosure has an improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, N1 comprises inosine and N4 comprises cytidine. In some embodiments, N1 comprises inosine and N4 comprises cytidine and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 comprises cytidine and N4 comprises inosine. In some embodiments, N2 comprise inosine and N3 comprises cytidine. In some embodiments, N2 comprises cytidine and N3 comprises inosine. In some embodiments, N1 comprises guanosine. In some embodiments, N2 comprises guanosine. In some embodiments, N1 comprises cytidine. In some embodiments, N2 comprises cytidine. In some embodiments, N1 and N2 comprise guanosine and N₃ and N₄ comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise guanosine. In some embodiments, N1 and N2 comprise inosine and N₃ and N₄ comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise inosine.

In some embodiments, the RLR agonists of the disclosure comprise the formula:

5′-(N₁-N₂-X1)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is the non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

wherein inosine, if present, base pairs with cytidine, and wherein N1 comprises inosine and N4 comprises cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N2 comprises inosine and N3 comprises cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine N₃ and N₄ comprise cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, Ni and N2 comprise guanosine and N₃ and N₄ comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine, X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine and no guanosine nucleosides. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine, X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise guanosine, and X1 and X2 each comprise inosine and no guanosine nucleosides. In some aspects, the RLR agonist of the disclosure has an improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is the non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

wherein inosine, if present, base pairs with cytidine, and wherein N1 and N2 comprise inosine and N₃ and N₄ comprise cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine and N₃ and N₄ comprise cytidine, X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise inosine and N₃ and N₄ comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine and N3 and N4 comprise cytidine, X1 and X2 each comprise at least one inosine, and X1 and X2 are each 12 nucleotides in length. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise inosine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine and N₃ and N₄ comprise cytidine, and X1 and X2 comprise inosine and no guanosine nucleosides. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise inosine, and X1 and X2 comprise inosine and no guanosine nucleosides. In some aspects, the RLR agonist of the disclosure has an improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments, the RLR agonists of the disclosure comprise the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein X1 and X2 are each 12 nucleotides and comprise 1, 2, 3 or 4 inosine nucleosides. In some embodiments, X1 and X2 are each 13 nucleotides and comprise 1, 2, 3, 4 or 5 inosine nucleosides. In some embodiments, X1 and X2 are each 14 nucleotides and comprise 1, 2, 3, 4, 5 or 6 inosine nucleosides. In some embodiments, X1 and X2 are each 15 nucleotides and comprise 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides and each comprise 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides and comprise at least 10%, 20%, 30% or 40% inosine nucleosides. In some aspects, the RLR agonist of the disclosure has an improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

-   -   (N₁-N₂-X₁) comprises a first polynucleotide comprising linked         nucleotides N₁, N₂ and X₁,     -   (ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising         linked nucleotides X₂, N₃ and N₄;     -   (iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide         comprising a nucleoside selected from the group consisting of:         adenosine, guanosine, cytidine, 5-methyluridine, uridine and         inosine;     -   (iv) N₁ base pairs with N₄;     -   (v) N₂ base pairs with N₃;     -   (vi) N₂ comprises a 5′ diphosphate or triphosphate moiety, or         derivative or analog thereof;     -   (vii) X₁ comprises a sequence motif [AUCN₅]_(x), wherein N₅ is         comprises guanosine or inosine, wherein x is an integer whose         value indicates the number of sequence motifs, and wherein x=3         or 4;     -   (viii) X₂ comprises a sequence motif [CN₆AU]_(y), wherein N6         comprises guanosine or inosine, wherein y is an integer whose         value indicates the number of sequence motifs, and wherein y=3         or 4;     -   (ix) L is a linker that operably links the first polynucleotide         and the second polynucleotide,

optionally, wherein at least one of N1, N2, N3, and N4 is inosine, and wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA.

In some embodiments, N5 comprises inosine and N6 comprises inosine. In some embodiments, N5 comprises guanosine and N6 comprises inosine. In some embodiments, N5 comprises inosine and N6 comprises guanosine. In some embodiments, N5 comprises guanosine (G) and N6 comprises guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine (I) and N4 comprises cytidine (C). In some embodiments, N2 comprises inosine (I) and N3 comprises cytidine (C). In some embodiments, N3 comprises inosine (I) and N2 comprises cytidine (C). In some embodiments, N4 comprises inosine (I) and N1 comprises cytidine (C). In some embodiments, N1 comprises guanosine (G). In some embodiments, N2 comprises guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G) and N₃ and N₄ comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N₃ and N₄ comprise guanosine (G). In some embodiments, N1 and N2 comprise inosine (I) and N₃ and N₄ comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N₃ and N₄ comprise inosine (I). In some aspects, the RLR agonist of the disclosure has an improved biological activity, wherein the improved biological activity is an increase in RLR-mediated cytokine production; an increase in RLR-mediated expression of interferon-stimulated genes; an increase in RLR-mediated intracellular signaling; an increase in binding affinity to RLRs; and a combination of any of the foregoing.

In some embodiments of the RLR agonist of the disclosure comprises a linker, wherein the linker is a nucleotide linker or a non-nucleotide linker. In some embodiments, the linker is a non-nucleotide linker. In some embodiments, the linker is a nucleotide linker. In some embodiments, the nucleotide linker comprises a tetraloop, wherein the nucleotide sequence of the tetraloop is selected from the group consisting of:

-   -   (a) UNCG, wherein N=A, C, G, or U;     -   (b) GNRA, wherein N=A, C, G, or U, and wherein R=A or G;     -   (c) ANYA, wherein N=A, C, G, or U, and wherein Y=C or T;     -   (d) CUYG, wherein Y=C or T;     -   (e) UMAC, wherein M=A or C; and     -   (f) CUUG.

In some embodiments, the sequence of the tetraloop is UUCG. In some embodiments, the sequence of the tetraloop is GAUC.

In some embodiments, the RLR agonist of the disclosure comprises a nucleotide linker, wherein the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

In some embodiments, the RLR agonist of the disclosure comprises a non-nucleotide linker, wherein the non-nucleotide linker is selected from the group consisting of:

-   -   (a) an ethylene glycol linker; and     -   (b) an alkyl linker.

In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

In some embodiments, the RLR agonist of the disclosure comprises a 5′ diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5′ triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog thereof comprises a phosphate bioisostere is selected from: a phosphonate, a thiophosphonate, a phosphorothioate, a sulfate, a sulfonate, a sulfamate, a thiazolidinone, a carboxylate, a malonate, a boronic acid, a benzoxaborole, a boranophosphate, a squaramide.

In some embodiments, the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkages or to the polynucleotide backbone.

In some embodiments, the RLR agonist of the disclosure exhibits one or more of the following properties:

-   -   (a) specifically binds to one or more RLRs (e.g. RIG-1, MDA5         and/or LGP2);     -   (b) increases RLR-mediated cytokine production;     -   (c) increases RLR-mediated expression of interferon-stimulated         genes (ISGs);     -   (d) increases RLR-dependent intracellular signaling;     -   (e) increases stability of the duplex;     -   (f) increases binding affinity to RLRs;     -   (g) decreases off-target binding;     -   (h) increases biological half-life;     -   (i) increases biodistribution and bioavailability;     -   (j) increases and/or enhances uptake into cells and/or tissues;     -   (k) decreases immunogenicity; and     -   (l) a combination of any of (a)-(k).

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁,

(ii) (X2-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is a linker that operably links the first polynucleotide and the second polynucleotide,

wherein N1 and N2 each comprise guanosine, wherein N₃ and N₄ each comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, wherein X1 and X2 each comprise at least one inosine nucleoside, wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA, and wherein L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is a linker that operably links the first polynucleotide and the second polynucleotide,

wherein N1 comprises inosine and N2 comprise guanosine, wherein N3 and N4 each comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, wherein X1 and X2 each comprise at least one inosine nucleoside, wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA, and wherein L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is a linker that operably links the first polynucleotide and the second polynucleotide,

wherein N1 and N2 comprise inosine and N3 and N4 comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, wherein X1 and X2 each comprise at least one inosine nucleoside, wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA, and wherein L comprises a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is UUCG.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a non-nucleotide linker, and wherein the agonist comprises the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is the non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

wherein N1 and N2 comprise guanosine, wherein N3 and N4 comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, and wherein the non-nucleotide linker is a C9 alkyl linker.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a non-nucleotide linker, and wherein the agonist comprises the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is the non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide,

wherein N1 and N2 comprise guanosine, wherein N3 and N4 comprise cytidine, wherein X1 and X2 are each 12 nucleotides in length, and wherein the non-nucleotide linker is a hexaethylene glycol linker.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLR, wherein the 5′ most nucleotide of the agonist comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, and wherein the agonist comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of:

(i) SEQ ID NO: 37 and 68, respectively;

(ii) SEQ ID NO: 38 and 69, respectively;

(iii) SEQ ID NO: 39 and 70, respectively;

(iv) SEQ ID NO: 40 and 71, respectively;

(v) SEQ ID NO: 41 and 72, respectively;

(vi) SEQ ID NO: 42 and 73, respectively;

(vii) SEQ ID NO: 43 and 74, respectively;

(viii) SEQ ID NO: 44 and 75, respectively;

(ix) SEQ ID NO: 45 and 76, respectively;

(x) SEQ ID NO: 46 and 77, respectively;

(xi) SEQ ID NO: 47 and 78, respectively;

(xii) SEQ ID NO: 48 and 79, respectively;

(xiii) SEQ ID NO: 49 and 80, respectively;

(xiv) SEQ ID NO: 50 and 81, respectively;

(xv) SEQ ID NO: 51 and 82, respectively;

(xvi) SEQ ID NO: 52 and 83, respectively;

(xvii) SEQ ID NO: 53 and 84, respectively;

(xviii) SEQ ID NO: 54 and 85, respectively;

(xix) SEQ ID NO: 55 and 86, respectively;

(xx) SEQ ID NO: 56 and 87, respectively;

(xxi) SEQ ID NO: 57 and 88, respectively;

(xxii) SEQ ID NO: 58 and 89, respectively;

(xxiii) SEQ ID NO: 59 and 89, respectively;

(xxiv) SEQ ID NO: 60 and 90, respectively;

(xxv) SEQ ID NO: 61 and 91, respectively;

(xxvi) SEQ ID NO: 62 and 92, respectively;

(xxvii) SEQ ID NO: 63 and 91, respectively;

(xxviii) SEQ ID NO: 64 and 93, respectively;

(xxix) SEQ ID NO: 65 and 94, respectively;

(xxx) SEQ ID NO: 66 and 95, respectively;

(xxxi) SEQ ID NO: 67 and 96, respectively; and

(xxxii) SEQ ID NO: 63 and 97, respectively.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, and wherein the agonist comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23 and 25.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, wherein the agonist comprises the formula 5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein (N₁-N₂-X₁) comprises a first polynucleotide and (X₂-N₃-N₄) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of:

(i) SEQ ID NO: 58 and 89, respectively;

(ii) SEQ ID NO: 59 and 89, respectively; and

(iii) SEQ ID NO: 61 and 91, respectively.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a non-nucleotide linker, wherein the agonist comprises the formula 5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein (N₁-N₂-X₁) comprises a first polynucleotide and (X₂-N₃-N₄) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of:

(i) SEQ ID NO: 37 and 68, respectively;

(ii) SEQ ID NO: 38 and 69, respectively;

(iii) SEQ ID NO: 39 and 70, respectively;

(iv) SEQ ID NO: 40 and 71, respectively;

(v) SEQ ID NO: 41 and 72, respectively;

(vi) SEQ ID NO: 42 and 73, respectively;

(vii) SEQ ID NO: 43 and 74, respectively;

(viii) SEQ ID NO: 44 and 75, respectively;

(ix) SEQ ID NO: 45 and 76, respectively;

(x) SEQ ID NO: 46 and 77, respectively;

(xi) SEQ ID NO: 47 and 78, respectively;

(xii) SEQ ID NO: 48 and 79, respectively;

(xiii) SEQ ID NO: 49 and 80, respectively;

(xiv) SEQ ID NO: 50 and 81, respectively;

(xv) SEQ ID NO: 51 and 82, respectively;

(xvi) SEQ ID NO: 52 and 83, respectively;

(xvii) SEQ ID NO: 53 and 84, respectively;

(xviii) SEQ ID NO: 54 and 85, respectively;

(xix) SEQ ID NO: 55 and 86, respectively;

(xx) SEQ ID NO: 56 and 87, respectively;

(xxi) SEQ ID NO: 57 and 88, respectively;

(xxii) SEQ ID NO: 58 and 89, respectively;

(xxiii) SEQ ID NO: 59 and 89, respectively;

(xxiv) SEQ ID NO: 60 and 90, respectively;

(xxv) SEQ ID NO: 61 and 91, respectively;

(xxvi) SEQ ID NO: 62 and 92, respectively;

(xxvii) SEQ ID NO: 63 and 91, respectively;

(xxviii) SEQ ID NO: 64 and 93, respectively;

(xxix) SEQ ID NO: 65 and 94, respectively;

(xxx) SEQ ID NO: 66 and 95, respectively;

(xxxi) SEQ ID NO: 67 and 96, respectively; and

(xxxii) SEQ ID NO: 63 and 97, respectively.

In some embodiments of the RLR agonists provided by the disclosure, the nucleotide sequence comprising the RLR agonist is not complementary to a genomic DNA sequence or mRNA sequence, wherein the RLR agonist does not participate in RNA interference, and wherein the RLR agonist does not silence gene expression.

In some aspects, the disclosure provides a pharmaceutical composition for stimulating an immune response, treating or delaying progression of a cancer, or reducing or inhibiting tumor growth in a subject in need thereof, comprising an RLR agonist provided by the disclosure, and a pharmaceutically acceptable carrier. In some embodiments, the RLR agonist is formulated in a polyethylenimine (PEI) carrier. In some embodiments, the PEI carrier is JetPEI®.

In some aspects, the disclosure provides a method to increase RLR-mediated production of one or more cytokines in a cell, the method comprising contacting the cell with an RLR agonist provided by the disclosure, wherein the RLR agonist increases RLR-mediated cytokine production in a cell. In some embodiments, the RLR agonist increases RLR-mediated type I interferon (e.g., IFN-α, IFN-β) production in a cell. In some embodiments, the RLR agonist increases RLR-mediated IL-1β production in a cell. In some embodiments, the RLR agonist increases RLR-mediated IP-10 production in a cell. In some embodiments, the RLR agonist increases RLR-mediated IL-6, IL-12p70, MCP-1 and/or MIP-1β production in a cell.

In some aspects, the disclosure provides a method to increase RLR-mediated expression of one or more interferon-stimulated genes in a cell, the method comprising contacting the cell with an RLR agonist provided by the disclosure, wherein the agonist increases RLR-mediated expression of one or more interferon-stimulated genes in a cell.

In some aspects, the disclosure provides method to increase RLR-dependent intracellular signaling in a cell, the method comprising contacting the cell with an RLR agonist provided by the disclosure, wherein the agonist increases RLR-dependent intracellular signaling.

In some aspects, the disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the disclosure.

In some aspects, the disclosure provides a method of treating or delaying progression of a cancer in a subject, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the disclosure.

In some aspects, the disclosure provides a method of reducing or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the disclosure.

In some aspects, the disclosure provides a method for stimulating an immune response, treating or delaying progression of a cancer, or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist or a pharmaceutical composition provided by the disclosure, wherein the agonist, or the pharmaceutical composition, increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-stimulated genes in a cell, and or increases RLR-dependent intracellular signaling in a cell, thereby stimulating the immune response, treating or delaying progression of the cancer, or inhibiting growth of the tumor.

In some embodiments of the methods provided by the disclosure, an RLR agonist or pharmaceutical composition provided by the disclosure is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cell death-inducing agent, an opsonizing agent (e.g., an opsonizing antibody) a cytotoxic agent, an immune-based therapy, a cytokine, an activator or agonist of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, a cellular immunotherapy, or a combination thereof.

In some embodiments, an RLR agonist or pharmaceutical composition provided by the disclosure is administered preceding or subsequent to administration of the one or more additional therapeutic agents or wherein the one or more additional therapeutic agents is administered concurrently with, preceding or subsequent to the administration of the agonist or pharmaceutical composition.

In some embodiments, the one or more additional therapeutic agents is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, a Toll-like receptor 9 (TLR9) agonist.

In some embodiments, the one or more additional therapeutic agents is an agonist comprising an polypeptide (e.g., antibody, or antigen binding portion thereof) that specifically binds to CD137 (4-1BB).

In some embodiments, the one or more additional therapeutic agents is an agonist comprising an polypeptide (e.g., antibody, or antigen binding portion thereof) that specifically binds to CD134 (OX40).

In some embodiments, the one or more additional therapeutic agents is a PD-1/PD-L1 antagonist. In some embodiments, the PD-1/PD-L1 antagonist is selected from the group consisting of: PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1/PD-L1 antagonist is selected from the group consisting of: FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559.

In some embodiments, the one or more additional therapeutic agents is a TIM-3 antagonist.

In some embodiments, the one or more additional therapeutic agents is a VISTA antagonist.

In some embodiments, the one or more additional therapeutic agents is an adenosine A2AR antagonist.

In some embodiments, the one or more additional therapeutic agents is a B7-H3 antagonist.

In some embodiments, the one or more additional therapeutic agents is a B7-H4 antagonist.

In some embodiments, the one or more additional therapeutic agents is a BTLA antagonist.

In some embodiments, the one or more additional therapeutic agents is a CTLA-4 antagonist.

In some embodiments, the one or more additional therapeutic agents is a IDO antagonist.

In some embodiments, the one or more additional therapeutic agents is a KIR antagonist.

In some embodiments, the one or more additional therapeutic agents is a LAG-3 antagonist.

In some embodiments, the one or more additional therapeutic agents is a Toll-like receptor 3 (TLR3) agonist. In some embodiments, the TLR3 agonist is polyinosinic:polycytidylic acid (poly I:C). In some embodiments, the TLR3 agonist is HILTONOL® (poly ICLC). In some embodiments, the TLR3 agonist is polyadenylic-polyuridylic acid (poly A:U). In some embodiments, the TLR3 agonist is RIBOXXIM® (RGIC®100). In some embodiments, the TLR3 agonist is RIBOXXON® (RGIC®50 bioconjugate). In some embodiments, the TLR3 agonist is RIBOXXOL (RGIC®50).

In some embodiments, the one or more additional therapeutic agents is a Toll-like receptor 7 (TLR7) agonist. In some embodiments, the TLR7 agonist is GS-9620 (Vesatolimod). In some embodiments, the TLR7 agonist is imiquimod (ALDARA™). In some embodiments, the TLR7 agonist is resiquimod (R-848).

In some embodiments, the one or more additional therapeutic agents is a Toll-like receptor 9 (TLR9) agonist. In some embodiments, the TLR9 agonist is a CpG oligodeoxynucleotide (CpG ODN). In some embodiments, the CpG ODN is a Class A CpG ODN (CpG-A ODN). In some embodiments, the CpG ODN is a Class B CpG ODN (CpG-B ODN). In some embodiments, the CpG ODN is a Class C CpG ODN (CpG-C ODN).

In some aspects, the disclosure provides a use of an RLR agonist or of a pharmaceutical composition provided by the disclosure, for stimulating an immune response, treating or delaying progression of a cancer, or inhibiting tumor growth in a subject in need thereof, optionally for use in combination with one or more additional therapeutic agents.

In some aspects, the disclosure provides a use of an RLR agonist or a pharmaceutical composition provided by the disclosure, in the manufacture of a medicament for stimulating an immune response, treating or delaying progression of a cancer, or inhibiting tumor growth in a subject in need thereof, optionally for use in combination with one or more additional therapeutic agents.

In some aspects, the disclosure provides a kit comprising an RLR agonist or a pharmaceutical composition provided by the disclosure and instructions for use in stimulating an immune response in a subject, or treating or delaying progression of a cancer, or inhibiting tumor growth in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents.

In some embodiments of a use or a kit provided by the disclosure, an RLR agonist or a pharmaceutical composition provided by the disclosure is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cell death-inducing agent, an opsonizing agent (e.g., an opsonizing antibody) a cytotoxic agent, an immune-based therapy, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, a cellular immunotherapy, or a combination thereof.

In some embodiments of a use or a kit provided by the disclosure, an RLR agonist or a pharmaceutical composition provided by the disclosure is administered preceding or subsequent to administration of the one or more additional therapeutic agents or wherein the one or more additional therapeutic agents is administered concurrently with, preceding or subsequent to the administration of the agonist or pharmaceutical composition.

In some embodiments of a use or a kit provided by the disclosure, the one or more additional therapeutic agents is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a Toll-like receptor 3 (TLR3) agonist, a Toll-like receptor 7 (TLR7) agonist, a Toll-like receptor 9 (TLR9) agonist.

In some embodiments of a use or kit provided by the disclosure, the one or more additional therapeutic agents is an agonist comprising an polypeptide (e.g., antibody, or antigen binding portion thereof) that specifically binds to CD137 (4-1BB).

In some embodiments of a use or kit provided by the disclosure, the disclosure the one or more additional therapeutic agents is an agonist comprising an polypeptide (e.g., antibody, or antigen binding portion thereof) that specifically binds to CD134 (OX40).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 provides a bar graph depicting the quantification of IFN-a secretion from human PBMC treated with 3 different concentrations of RIG-I-like receptor agonists comprising various modifications.

DETAILED DESCRIPTION Overview

The RIG-I-like receptors (RLRs) are a family of cytosolic pattern recognition receptors that are essential for detecting viral RNA and initiating the innate immune response. The RLR family includes three members: Retinoic acid-inducible gene I (RIG-I), Melanoma differentiation-associated gene 5 (MDA5), and Laboratory of genetics and physiology 2 (LGP2). These receptors are expressed in both immune and non-immune cell types and regulate signaling pathways that promote the IRF3-, IRF7-dependent expression of type I and type III interferons (IFNs), and the NF-kappa B-dependent expression of pro-inflammatory cytokines.

All three RLR family receptors have a DExD/H box RNA helicase domain with ATPase activity. This domain along with the adjacent C-terminal domain is required for RNA binding. In addition, the C-terminal domains of RIG-I and LGP2 have been shown to act as repressor domains, ensuring that the receptors remain in an inactive conformation until they are bound by an activating RNA.

The present disclosure provides RLR agonists comprising synthetic RNA molecules that fold to form a duplexed, dsRNA and that comprise one or more sequence motifs that provides one or more improved biological activities.

Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified. In the case of direct conflict with a term used in a parent provisional patent application, the term used in the instant application shall control.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

About: As used herein, the term “about” (alternatively “approximately”) will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, “about” will mean up to plus or minus 10% of the particular value.

Agonist: As used herein, the term “agonist” is used in its broadest sense and encompasses any molecule or compound that partially or fully promotes, induces, increases, and/or activates a biological activity of a native polypeptide disclosed herein. Agonist molecules according to the disclosure may include nucleic acids (e.g., oligonucleotides, polynucleotides), antibodies or antigen-binding fragments, fragments or amino acid sequence variants of native polypeptides, peptides, oligonucleotides, lipids, carbohydrates, and small organic molecules. In some embodiments, activation in the presence of the agonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured with a negative control under comparable conditions. Also disclosed herein, are methods of identifying agonists suitable for use in the methods of the disclosure. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio© systems, fluorescence polarization (FP) assay, and radioimmunoassay (RIA). These assays determine the ability of an agonist to bind the polypeptide of interest (e.g., a receptor or ligand) and therefore indicate the ability of the agonist to promote, increase or activate the activity of the polypeptide. Efficacy of an agonist can also be determined using functional assays, such as the ability of an agonist to activate or promote the function of the polypeptide. For example, a functional assay may comprise contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The potency of an agonist is usually defined by its EC₅₀ value (concentration required to activate 50% of the agonist response). The lower the EC₅₀ value the greater the potency of the agonist and the lower the concentration that is required to activate the maximum biological response.

Ameliorating: As used herein, the term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., cancer, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

Amino acid: As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

Amino acid substitution: As used herein, an “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different “replacement” amino acid residue. An “amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger “peptide insertions,” can also be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

Base Composition: As used herein, the term “base composition” refers to the proportion of the total nucleotides of a nucleic acid (e.g., an RNA) consisting of guanine (or hypoxanthine)+cytosine and/or uracil (or thymine)+adenine nucleobases.

Base Pair: As used herein, the term “base pair” refers to two nucleobases on opposite complementary polynucleotide strands, or regions of the same strand, that interact via the formation of specific hydrogen bonds. As used herein, the term “Watson-Crick base pairing”, used interchangeably with “complementary base pairing”, refers to a set of base pairing rules, wherein a purine always binds with a pyrimidine such that the nucleobase adenine (A) forms a complementary base pair with thymine (T) and guanine (G) forms a complementary base pair with cytosine (C) in DNA molecules. In RNA molecules, thymine is replaced by uracil (U), which, similar to thymine (T), forms a complementary base pair with adenine (A). The complementary base pairs are bound together by hydrogen bonds and the number of hydrogen bonds differs between base pairs. As in known in the art, guanine (G)-cytosine (C) base pairs are bound by three (3) hydrogen bonds and adenine (A)-thymine (T) or uracil (U) base pairs are bound by two (2) hydrogen bonds.

Base pairing interactions that do not follow these rules can occur in natural, non-natural, and synthetic nucleic acids and are referred to herein as “non-Watson-Crick base pairing” or alternatively “non-canonical base pairing”. A “wobble base pair” is a pairing between two nucleobases in RNA molecules that does not follow Watson-Crick base pair rules. For example, inosine is a nucleoside that is structurally similar to guanosine, but is missing the 2-amino group. Inosine is able to form two hydrogen bonds with each of the four natural nucleobases (Oda et al., (1991) Nucleic Acids Res 19:5263-5267) and it is often used by researchers as a “universal” base, meaning that it can base pair with all the naturally-occurring or canonical bases. The four main wobble base pairs are the guanine-uracil (G-U) base pair, the hypoxanthine-uracil (I-U) base pair, the hypoxanthine-adenine (I-A) base pair, and the hypoxanthine-cytosine (I-C) base pair. In order to maintain consistency of nucleic acid nomenclature, “I” is used for hypoxanthine because hypoxanthine is the nucleobase of inosine; nomenclature otherwise follows the names of nucleobases and their corresponding nucleosides (e.g., “G” for both guanine and guanosine—as well as for deoxyguanosine). The thermodynamic stability of a wobble base pair is comparable to that of a Watson-Crick base pair. Wobble base pairs play a role in the formation of secondary structure in RNA molecules.

In one aspect, the disclosure provides synthetic RNA molecules that agonize or activate one or more RIG-I-like receptors (RLRs), wherein inosine can only be inserted at positions where it will base pair with cytidine (I-C base pair); that is, inosine can be substituted for guanosine but cannot be substituted for the other nucleosides.

Biologically active: As used herein, the phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active and thus have “biological activity”. In particular embodiments, where a nucleic acid is biologically active, a portion of that nucleic acid that shares at least one biological activity of the whole nucleic acid is typically referred to as a “biologically active” portion.

Covalently linked: As used herein, the term “covalently linked” (alternatively “conjugated”, “linked,” “attached,” “fused”, or “tethered”), when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, by whatever means including chemical conjugation, recombinant techniques or enzymatic activity, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.

Complementary: As used herein, the term “complementary” or “complementarity” refers to a relationship between the sequence of nucleotides comprising two polynucleotide strands, or regions of the same polynucleotide strand, and the formation of a duplex comprising the strands or regions, wherein the extent of consecutive base pairing between the two strands or regions is sufficient for the generation of a duplex structure. It is known that adenine (A) forms specific hydrogen bonds, or “base pairs”, with thymine (T) or uracil (U). Similarly, it is known that a cytosine (C) base pairs with guanine (G). It is also known that non-canonical nucleobases (e.g., inosine) can hydrogen bond with natural bases. A sequence of nucleotides comprising a first strand of a polynucleotide, or a region, portion or fragment thereof, is said to be “sufficiently complementary” to a sequence of nucleotides comprising a second strand of the same or a different nucleic acid, or a region, portion, or fragment thereof, if, when the first and second strands are arranged in an antiparallel fashion, the extent of base pairing between the two strands maintains the duplex structure under the conditions in which the duplex structure is used (e.g., physiological conditions in a cell). It should be understood that complementary strands or regions of polynucleotides can include some base pairs that are non-complementary. Complementarity may be “partial,” in which only some of the nucleobases comprising the polynucleotide are matched according to base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. Although the degree of complementarity between polynucleotide strands or regions has significant effects on the efficiency and strength of hybridization between the strands or regions, it is not required for two complementary polynucleotides to base pair at every nucleotide position. In some embodiments, a first polynucleotide is 100% or “fully” complementary to a second polynucleotide and thus forms a base pair at every nucleotide position. In some embodiments, a first polynucleotide is not 100% complementary (e.g., is 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches.

Contacting: As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a cell with an agent (e.g. an RNA, a lipid nanoparticle composition, or other pharmaceutical composition of the disclosure) means that the cell and the agent are made to share a physical connection. Methods of contacting cells with external entities both in vivo, in vitro, and ex vivo are well known in the biological arts. In exemplary embodiments of the disclosure, the step of contacting a mammalian cell with a composition (e.g., an isolated RNA, nanoparticle, or pharmaceutical composition of the disclosure) is performed in vivo. For example, contacting a lipid nanoparticle composition and a cell (for example, a mammalian cell) which may be disposed within an organism (e.g., a mammal) may be performed by any suitable administration route (e.g., parenteral administration to the organism, including intravenous, intramuscular, intradermal, and subcutaneous administration). For a cell present in vitro, a composition (e.g., a lipid nanoparticle or an isolated RNA) and a cell may be contacted, for example, by adding the composition to the culture medium of the cell and may involve or result in transfection. Moreover, more than one cell may be contacted by an agent.

Denaturation: As used herein, the term “denaturation” refers to the process by which the hydrogen bonding between base paired nucleotides in a nucleic acid is disrupted, resulting in the loss of secondary and/or tertiary nucleic acid structure (e.g. the separation of previously annealed strands). Denaturation can occur by the application of an external substance, energy, or biochemical process to a nucleic acid.

Antigen presenting cell: The term “antigen presenting cell” or “APC” is a cell that displays foreign antigen complexed with MHC on its surface. T cells recognize this complex using T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens either by phagocytosis or by receptor-mediated endocytosis.

Apoptosis: As used herein, the term “apoptosis” refers to the process of programmed cell death that occurs in multicellular organisms (e.g. humans). The highly-regulated biochemical and molecular events that result in apoptosis can lead to observable and characteristic morphological changes to a cell, including membrane blebbing, cell volume shrinkage, chromosomal DNA condensation and fragmentation, and mRNA decay. A common method to identify cells, including T cells, undergoing apoptosis is to expose cells to a fluorophore-conjugated protein (Annexin V). Annexin V is commonly used to detect apoptotic cells by its ability to bind to phosphatidylserine on the outer leaflet of the plasma membrane, which is an early indicator that the cell is undergoing the process of apoptosis.

Blunt-end: As used herein, the term “blunt-end” “blunt-ended” refers to the structure of an end of a duplexed or double-stranded nucleic acid, wherein both complementary strands comprising the duplex terminate, at least at one end, in a base pair. Hence, neither strand comprising the duplex extends further from the end than the other.

Cancer antigen: As used herein, “cancer antigen” refers to (i) tumor-specific antigens, such as neoantigens, (ii) tumor-associated antigens, (iii) cells that express tumor-specific antigens, (iv) cells that express tumor-associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.

Carcinoma: As used herein, the term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The RIG-I-like receptor (RLR) agonists described herein can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

Cytotoxic T lymphocyte (CTL) response: As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+T cells.

Duplex: As used herein, the term “duplex” refers to a structure formed by complementary strands of a double-stranded polynucleotide, or complementary regions of a single-stranded polynucleotide that folds back on itself. The duplex structure of a nucleic acid arises as a consequence of complementary nucleotide sequences being bound together, or hybridizing, by base pairing interactions.

EC₅₀: As used herein, the term “EC₅₀” refers to the concentration of an agonist which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline.

Effective dose: As used herein, the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect.

Hairpin RNA: As used herein, the term “hairpin RNA” or “RNA hairpin” refers to a self-complementary RNA comprising a double-stranded RNA (dsRNA) stem comprised of complementary nucleotide strands that base pair to form a duplex that terminates at one end in a nucleotide linker comprising a loop of unpaired nucleotides (e.g., a tetraloop) comprising unpaired nucleotides or in a non-nucleotide linker comprising a flexible chemical moiety (e.g., ethylene glycol), either of which connects the complementary nucleotide strands. RNA hairpins may differ in the length of the stem, the size and/or composition of the loop and/or linker, the number of base pair mismatches within the stem, and in the actual nucleotide sequence. RNA hairpins may provide one or more functions, including, but not limited to, guiding the overall folding of an RNA molecule comprising the hairpin, determining interactions in a ribozyme, protecting messenger RNA (e.g., mRNA) from degradation, serving as a recognition motif or structure for RNA binding proteins and acting as a substrate for enzymatic reactions. Further description of RNA hairpin structures and functions can be found in Svoboda and Di Cara (2006) Cell Mol Life Sci 63(7-8):901-908, and references contained therein. In some embodiments, the stem regions of the hairpin RNAs comprising the RLR agonists provided by the disclosure terminate in a blunt end with a 5′ triphosphate or diphosphate.

In need: As used herein, a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising a RIG-I-like receptor agonist).

Linker: As used herein, the term “linker” (alternatively “tether” or “spacer”) refers to a moiety that covalently connects, attaches or couples two polynucleotide strands or regions together. As used herein, a linker comprising nucleotides is referred to as a “nucleotide linker” (e.g. a tetraloop). As used herein, the term “non-nucleotide linker” refers to a linker comprising a chemical moiety and that does not comprise a nucleotide. Non-limiting examples of non-nucleotide linkers include linkers comprising ethylene glycol (e.g. hexaethylene glycol), alkyl chains (e.g. C9 alkyl linker), and stilbene diether. Further description of linkers can be found in Paredes et al., (2011) Methods 54:251-259, which is incorporated herein by reference in its entirety.

LGP2: As used herein, the term “LGP2” refers to the Laboratory of Genetics and Physiology 2 polypeptide, a specific member of the RIG-I-like receptor family and is encoded by the DHX58 gene in humans. Alternative names and acronyms for LGP2 in the art include DHX58, D11LGP2, D11Igp2e, and RLR-3. An exemplary amino acid sequence of full-length human LGP2 is set forth in Table 4 (SEQ ID NO: 100) and here:

(NCBP Accession Number: NP_077024.2) MELRSYQWEVIMPALEGKNIIIWLPTGAGKTRAAAYVAKRHLETVDGAKV VVLVNRVHLVTQHGEEFRRMLDGRWTVTTLSGDMGPRAGFGHLARCHDLL ICTAELLQMALTSPEEEEHVELTVFSLIVVDECHHTHKDTVYNVIMSQYL ELKLQRAQPLPQVLGLTASPGTGGASKLDGAINHVLQLCANLDTWCIMSP QNCCPQLQEHSQQPCKQYNLCHRRSQDPFGDLLKKLMDQIHDHLEMPELS RKFGTQMYEQQVVKLSEAAALAGLQEQRVYALHLRRYNDALLIHDTVRAV DALAALQDFYHREHVTKTQILCAERRLLALFDDRKNELAHLATHGPENPK LEMLEKILQRQFSSSNSPRGIIFTRTRQSAHSLLLWLQQQQGLQTVDIRA QLLIGAGNSSQSTHMTQRDQQEVIQKFQDGTLNLLVATSVAEEGLDIPHC NVVVRYGLLTNEISMVQARGRARADQSVYAFVATEGSRELKRELINEALE TLMEQAVAAVQKMDQAEYQAKIRDLQQAALTKRAAQAAQRENQRQQFPVE HVQLLCINCMVAVGHGSDLRKVEGTHHVNVNPNFSNYYNVSRDPVVINKV FKDWKPGGVISCRNCGEVWGLQMIYKSVKLPVLKVRSMLLETPQGRIQAK KWSRVPFSVPDFDFLQHCAENLSDLSLD

Local administration: As used herein, “local administration” or “local delivery,” refers to delivery that does not rely upon transport of the composition or agent to its intended target tissue or site via the vascular system. For example, the composition may be delivered by injection or implantation of the composition or agent or by injection or implantation of a device containing the composition or agent. Following local administration in the vicinity of a target tissue or site, the composition or agent, or one or more components thereof, may diffuse to the intended target tissue or site.

MDA5: As used herein, the term “MDA5” refers to the Melanoma Differentiation-Associated Protein 5 polypeptide, a specific member of the RIG-I-like receptor family and is encoded by the IFIH1 gene in humans. Alternative names and acronyms for MDA5 in the art include AGS7, Hlcd, IDDM19, MDA-5, RLR-2, SGMRT1, and interferon induced with helicase C domain 1. An exemplary amino acid sequence of full-length human MDA5 is set forth in Table 4 (SEQ ID NO: 99) and here:

(NCBI Accession Number: NP_071451.2) MSNGYSTDENFRYLISCFRARVKMYIQVEPVLDYLTFLPAEVKEQIQRTV ATSGNMQAVELLLSTLEKGVWHLGWTREFVEALRRTGSPLAARYMNPELT DLPSPSFENAHDEYLQLLNLLQPTLVDKLLVRDVLDKCMEEELLTIEDRN RIAAAENNGNESGVRELLKRIVQKENWFSAFLNVLRQTGNNELVQELTGS DCSESNAEIENLSQVDGPQVEEQLLSTTVQPNLEKEVWGMENNSSESSFA DSSVVSESDTSLAEGSVSCLDESLGHNSNMGSDSGTMGSDSDEENVAARA SPEPELQLRPYQMEVAQPALEGKNIIICLPTGSGKTRVAVYIAKDHLDKK KKASEPGKVIVLVNKVLLVEQLFRKEFQPFLKKWYRVIGLSGDTQLKISF PEVVKSCDIIISTAQILENSLLNLENGEDAGVQLSDFSLIIIDECHHTNK EAVYNNIMRHYLMQKLKNNRLKKENKPVIPLPQILGLTASPGVGGATKQA KAEEHILKLCANLDAFTIKTVKENLDQLKNQIQEPCKKFAIADATREDPF KEKLLEIMTRIQTYCQMSPMSDFGTQPYEQWAIQMEKKAAKEGNRKERVC AEHLRKYNEALQINDTIRMIDAYTHLETFYNEEKDKKFAVIEDDSDEGGD DEYCDGDEDEDDLKKPLKLDETDRFLMTLFFENNKMLKRLAENPEYENEK LTKLRNTIMEQYTRTEESARGIIFTKTRQSAYALSQWITENEKFAEVGVK AHHLIGAGHSSEFKPMTQNEQKEVISKFRTGKINLLIATTVAEEGLDIKE CNIVIRYGLVTNEIAMVQARGRARADESTYVLVAHSGSGVIEHETVNDFR EKMMYKAIHCVQNMKPEEYAHKILELQMQSIMEKKMKTKRNIAKHYKNNP SLITFLCKNCSVLACSGEDIHVIEKMHHVNMTPEFKELYIVRENKALQKK CADYQINGEIICKCGQAWGTMMVHKGLDLPCLKIRNFVVVFKNNSTKKQY KKWVELPITFPNLDYSECCLFSDED

Modified: As used herein “modified” or “modification” refers to a changed state or change in structure resulting from a modification of a polynucleotide, e.g., RNA. Polynucleotides may be modified in various ways including chemically, structurally, and/or functionally. For example, the RNA molecules of the present disclosure may be modified by the incorporation of a non-natural base or a sequence motif, comprising a functional sequence or secondary structure, that provides a biological activity. In one embodiment, the RNA is modified by the introduction of non-natural or chemically-modified bases, nucleosides and/or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C.

Naturally-occurring: As used herein, the term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence, or components thereof such as amino acids or nucleotides, that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

Nucleic acid: As used herein, the term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers or oligomers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Polymers of nucleotides are referred to as “polynucleotides”. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

Polynucleotides used herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases. “Modified nucleosides” include, for example, as inosine and thymine, when the latter is found in or comprises RNA. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

Nucleic Acid Structure: As used herein, the term “nucleic acid structure” refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and/or sequence of nucleobases that comprise a nucleic acid (e.g. an RNA) and/or can refer to the two-dimensional or three-dimensional state of a nucleic acid. Accordingly, the term “RNA structure” refers to the arrangement or organization of atoms, chemical constituents, elements, motifs, and/or sequence of nucleobases comprising an RNA molecule (e.g. an mRNA) and/or can refer to the two-dimensional and/or three dimensional state of an RNA molecule. Nucleic acid structure can be further demarcated into four organizational categories referred to herein as “molecular structure”, “primary structure”, “secondary structure”, and “tertiary structure” based on increasing organizational complexity.

Nucleobase: As used herein, the term “nucleobase” (alternatively “nucleotide base” or “nitrogenous base”) refers to a purine or pyrimidine heterocyclic compound found in nucleic acids, including any derivatives or analogs of the naturally occurring purines and pyrimidines that confer improved properties (e.g. binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Adenine, cytosine, guanine, thymine, and uracil are the primary or canonical nucleobases predominately found in natural nucleic acids. Other natural, non-natural, non-canonical and/or synthetic nucleobases, can be incorporated into nucleic acids, such as those disclosed herein.

Nucleoside/Nucleotide: As used herein, the term “nucleoside” refers to a compound containing a sugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), or derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as “nucleobase”). As used herein, the term “nucleotide” refers to a nucleoside covalently linked to a phosphate group. As used herein, the term “ribonucleoside” refers to a nucleoside that comprise a ribose and a nucleobase (e.g., adenosine (A), cytidine (C), guanosine (G), 5-methyluridine (m⁵U), uridine (U), or inosine (I)).

Operably linked: As used herein, a nucleic acid, or fragment or portion thereof, such as a polynucleotide or oligonucleotide is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence, or fragment or portion thereof.

Polynucleotide/oligonucleotide: As used herein, the terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a single-stranded or double-stranded polymer or oligomer of nucleotides or nucleoside monomers consisting of naturally-occurring bases, sugars and intersugar (backbone) linkages. The terms “polynucleotide” and “oligonucleotide” also includes polymers and oligomers comprising non-naturally occurring bases, sugars and intersugar (backbone) linkages, or portions thereof, which function similarly. Polynucleotides are not limited to any particular length of nucleotide sequence, as the term “polynucleotides” encompasses polymeric forms of nucleotides of any length. Short polynucleotides are typically referred to in the art as “oligonucleotides”. In the context of the present disclosure, such modified or substituted polynucleotides and oligonucleotides are often preferred over native forms because the modification increases one or more desirable or beneficial biological properties or activities including, but not limited to, increased cytokine production, enhanced cellular uptake and/or increased stability in the presence of nucleases. In some embodiments, the agonists of the disclosure comprise polynucleotides and oligonucleotides that contain at least one region of modified nucleotides that confers one or more beneficial properties or increases biological activity (e.g., increased nuclease resistance, increased uptake into cells, increased duplex stability, increased binding affinity to a target polypeptide).

Palindromic sequence: As used herein, the term “palindromic sequence” (alternatively “palindrome”) refers to a sequence of nucleotides that is self-complementary; wherein the sequence of nucleotides in the 5′ to 3′ direction is the same as the sequence of nucleotides comprising the complementary strand, when read in the 5′ to 3′. For example, the sequence 5′-ACCTAGGT-3′ is a palindromic sequence because its complementary sequence, 3′-TGGATCCA-5′, when read in the 5′ to 3′ direction, is the same as the original sequence. In contrast, the sequence 5′-AGTGGCTG-3′ is not a palindromic sequence because its complementary sequence, 3′-TCACCGAC-5′, when read in the 5′ to 3′ direction, is not the same as the original sequence.

In one embodiment, the agonist is comprised of a first oligonucleotide, wherein the sequence of the first oligonucleotide is a palindromic sequence. In another embodiment, the agonist is comprised of a first oligonucleotide, wherein the first oligonucleotide comprises a palindromic sequence.

Palindromic sequences in preferred oligonucleotides of the invention preferably include both the 5′ end of the oligonucleotide and the 3′ end of the oligonucleotide, thus forming a blunt end. In one embodiment of the invention the oligonucleotide comprises a single palindromic sequence and in another more preferred embodiment of the invention the oligonucleotide comprises two complementary palindromes interrupted by an intervening sequence, spacer, or linker that connects the 2 palindromes within 1 or 2 different oligonucleotides so as to form a hairpin duplex with a blunt end.

Parenteral administration: As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion.

Patient: As used herein, the term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

Percent identity: As used herein, the term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausub el et al., infra).

One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and)(BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19).

Phosphate: The term “phosphate” as used herein means a salt or ester of phosphoric acid. Polyphosphates are salts or esters of polymeric oxyanions formed from tetrahedral PO₄ (phosphate) structural units linked together by sharing oxygen atoms. As used herein, the term “diphosphate” refers to a polyphosphate comprising two phosphate structural units. As used herein, the term “triphosphate” refers to a polyphosphate comprising three phosphate structural units. In some embodiments, the disclosure provides a RIG-I-like receptor agonist comprising a diphosphate moiety, or a derivative or analog thereof, linked to the 5′ terminus. In some embodiments, the disclosure provides a RIG-I-like receptor agonist comprising a triphosphate moiety, or a derivative or analog thereof, linked to the 5′ terminus. In some embodiments, the derivative or analog thereof is a phosphate bioisostere.

Phosphate bioisostere: As used herein, the term “phosphate bioisostere” (alternatively “phosphate mimic”) refers to chemical substituents or groups with similar physical or chemical properties to phosphate and which produce broadly similar biological properties to phosphate, including biphosphate and triphosphate moieties. In drug design, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structure. The use of bioisosteres is widespread in drug development and is used, for example, to reduce toxicity, change bioavailability, or modify the activity or metabolism of the parental or lead compound (see e.g., Rye and Baell (2005) Curr Med Chem 12(26):3127-3141; Elliot et al., (2012) MedChemCom 3(7):735-751, which are incorporated herein by reference in their entirety).

Polypeptide: As used herein, the terms “polypeptide,” “peptide”, and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

Preventing: As used herein, the term “preventing” when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

Purified: As used herein, the term “purified” or “isolated” as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample.

Reference ligand: As used herein, the term “reference ligand” (used interchangeably with “reference agonist”) or “reference molecule” refers to a RIG-I-like receptor ligand and is used to establish a relationship between itself and one or more distinct RIG-I-like receptor ligands, wherein the relationship is the relative agonistic effect of the reference ligand and the one or more distinct RIG-I-like receptor ligands. As used herein, the term connotes a RIG-I-like receptor ligand or agonist that is useful in a test or assay, such as those described herein, (e.g., an IFN induction assay), as a competitor, wherein the assay is useful for the discovery, identification or development, of one or more distinct agonists that bind to RIG-I-like receptors.

RIG-I: As used herein, the term “RIG-I” refers to the Retinoic Acid-Inducible Gene I polypeptide, a specific member of the RIG-I-like receptor family and is encoded by the DDX58 gene in humans. Alternative names and acronyms for RIG-I in the art include DEAD box polypeptide 58, RIGI, RLR-1, SGMRT2, and DEXD/H-box helicase 58. An exemplary amino acid sequence of full-length human RIG-I is set forth in Table 4 (SEQ ID NO: 98) and here:

(NCBI Accession Number: NP_055129.2) MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPWFREEEVQYIQAEKNNKGP MEAATLFLKFLLELQEEGWFRGFLDALDHAGYSGLYEAIESWDFKKIEKL EEYRLLLKRLQPEFKTRIIPTDIISDLSECLINQECEEILQICSTKGMMA GAEKLVECLLRSDKENWPKTLKLALEKERNKFSELWIVEKGIKDVETEDL EDKMETSDIQIFYQEDPECQNLSENSCPPSEVSDTNLYSPFKPRNYQLEL ALPAMKGKNTIICAPTGCGKTFVSLLICEHHLKKFPQGQKGKVVFFANQI PVYEQQKSVFSKYFERHGYRVTGISGATAENVPVEQIVENNDIIILTPQI LVNNLKKGTIPSLSIFTLMIFDECHNTSKQHPYNMIMFNYLDQKLGGSSG PLPQVIGLTASVGVGDAKNTDEALDYICKLCASLDASVIATVKHNLEELE QVVYKPQKFFRKVESRISDKFKYIIAQLMRDTESLAKRICKDLENLSQIQ NREFGTQKYEQWIVTVQKACMVFQMPDKDEESRICKALFLYTSHLRKYND ALIISEHARMKDALDYLKDFFSNVRAAGFDEIEQDLTQRFEEKLQELESV SRDPSNENPKLEDLCFILQEEYHLNPETITILFVKTRALVDALKNWIEGN PKLSFLKPGILTGRGKTNQNTGMTLPAQKCILDAFKASGDHNILIATSVA DEGIDIAQCNLVILYEYVGNVIKMIQTRGRGRARGSKCFLLTSNAGVIEK EQINMYKEKMMNDSILRLQTWDEAVFREKILHIQTHEKFIRDSQEKPKPV PDKENKKLLCRKCKALACYTADVRVIEECHYTVLGDAFKECFVSRPHPKP KQFSSFEKRAKIFCARQNCSHDWGIHVKYKTFEIPVIKIESFVVEDIATG VQTLYSKWKDFHFEKIPFDPAEMSK

RIG-I-like receptor: As used herein, the term “RIG-I-like receptor” (abbreviate as “RLR”) refers to any member of a family of DExD/H box RNA helicases that function as cytoplasmic pattern recognition sensors of pathogen-associated molecular patterns (PAMPs) typically found in viral RNA. Upon ligand binding, RLRs signal downstream transcription factor activation to drive type 1 interferon (IFN) production and antiviral gene expression that elicits an intracellular immune response to control virus infection. Three RLR members have been identified: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation associated factor 5), and LGP2 (laboratory of genetics and physiology 2 and a homolog of mouse D11lgp2) (Loo and Gale (2011) Immunity 34(5):680-692).

RIG-I-like receptor agonist: As used herein, the term “RIG-I-like receptor agonist” (used interchangeably with the term “RLR agonist”) refers to a nucleic acid (e.g., an RNA) that binds to RIG-I-like receptors (RLRs) and partially or fully promotes, induces, increases, and/or activates a biological activity, response, and/or downstream pathway(s) mediated by RLR signaling or other RLR-mediated function. Examples of RIG-I-receptor agonists are provided herein.

Stable RNA secondary structure: As used herein, the term “stable RNA secondary structure” refers to a structure, fold, or conformation adopted by an RNA molecule, or local segment or portion thereof, that is persistently maintained under physiological conditions and characterized by a low free energy state. Typical examples of stable RNA secondary structures include duplexes, hairpins, and stem-loops. Stable RNA secondary structures are known in the art to exhibit various biological activities. The term “stable” as used in reference to a polynucleotide duplex, means that the duplex remains hybridized, structured or annealed essentially exclusively in the form of a duplex under physiological conditions or under typical salt and temperature conditions used in nucleic acid diagnostic or therapeutic applications.

Subject: As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

T cell: The term “T cell” refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (a.k.a. TH cells or CD4+ T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (a.k.a TC cells, CD8+ T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Treg cells or suppressor T cells) and subtypes, including CD4+ FOXP3+ Treg cells, CD4+FOXP3-Treg cells, Tr1 cells, Th3 cells, and Treg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.

T cell activation: As used herein, the term “T cell activation” or “activation of T cells” refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell-based effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MEW), and the second by subsequent engagement of co-stimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

T cell-mediated response: As used herein, the term “T cell-mediated response” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

Tetraloop: As used herein, the term “tetraloop” refers to a type of four-base loop motif found in hairpin or stem-loop RNA secondary structures that cap duplexes at one end, linking the two strands comprising the duplex, and provide stability to the hairpin structure.

Therapeutic agent: As used herein, the term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

Therapeutically effective amount: As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., a synthetic RIG-I-like receptor agonist) that will elicit the desired biological or medical response, such as, for example, curing or at least partially arresting the condition or disease and its complications in a patient already suffering from the disease (e.g., an improvement in one or more symptoms of a cancer). Amounts effective for this use will depend on the severity of the disorder being treated and the general state of the patient's own immune system.

Treat: The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

Tumor microenvironment: As used herein, the term “tumor microenvironment” (alternatively “cancer microenvironment”; abbreviated TME) refers to the cellular environment or milieu in which the tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME. The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments, described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded thereby; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

RIG-I-Like Receptors and Their Ligands

The present disclosure provides synthetic RNA ligands that specifically bind to RIG-I-like receptors (RLRs) and agonize RLRs (RLR agonists). In some aspects, the disclosure provides RLR agonists that are useful for the treatment of cancer. In some aspects, the disclosure provides RLR agonists that are useful for the treatment of infectious disease. In some embodiments, the RLR agonists induce cytokine production. In some embodiments, the RLR agonists increase the number of CD8+ T cells in the tumor microenvironment. In some embodiments, the RLR agonists induce protective anti-tumor immunity

RIG-I-like receptors (RLRs) comprise a family of DExD/H box RNA helicases that function as cytosolic pattern recognition receptors (PRRs) that sense the presence of pathogenic agents via the recognition of pathogen-associated molecular patterns (PAMPs). In particular, the intracellular presence of non-self (e.g., viral) RNA is sensed by an infected cell via binding of the RNA to RLRs and results in the initiation and modulation of antiviral immunity. Like most viral RNAs, endogenous mRNA and RNA polymerase III transcripts are also 5′-triphosphorylated, but eukaryotic mRNAs possess a 5′ cap structure linked to a guanosine methylated at N7 that prevents RIG-I activation. These structural differences between viral and self RNAs, together with differences in intracellular localization, are thought to enable the effective function of RIG-I as a defense against viral infection by the preferential detection of viral RNA. The molecular recognition and binding of non-self RNA ligands to RLRs propagates specific intracellular signal events culminating in the activation of transcription factors that drive type 1 interferon (IFN) production and antiviral gene expression. The RLR-mediated induction of IFN and inflammatory cytokines production as well as antiviral gene expression elicits an immune response to control virus infection (Yoneyama et al., (2015) Curr Opin Immunol 32:48-53).

Three RLR family members have been identified: RIG-I (retinoic acid-inducible gene I)—the founding member and best characterized of RLR family, MDAS (melanoma differentiation associated factor 5), and LGP2 (laboratory of genetics and physiology 2 and a homolog of mouse D11lgp2). RIG-I is an important component of the innate immune system and plays a critical role in the defense against infection by RNA viruses. In contrast to the Toll-like receptors TLR3, TLR7, TLR8, and TLR9, that detect nucleic acids in the endosomes of a subset of immune cells, RIG-I is a cytosolic innate immune receptor that is expressed in all cell types (Kato et al., (2006) Nature 441(7089):101-105; Loo et al., (2008) J Virol 82(1):335-345). Two early studies independently established that RIG-I specifically detects and is activated by viral RNAs (Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006) Science 314(5801):997-1001).

High-resolution structures of RIG-I/ligand complexes have provided the molecular detail of RIG-I binding to RNA ligands, specifically to the activating ligand, double-stranded 5′-triphosphorylated RNA (ppp-dsRNA) (Civril et al., (2011) EMBO Reports 12(11):1127-1134; Jiang et al., (2011) Nature 479(7373):423-427; Kowalinski et al., (2011) Cell 147(2):423-435; Lu et al., (2010) Structure 18(8): 1032-1043; Luo et al., (2011) Cell 147(2) 409-422; Wang et al., (2010) Nature Structural & Molecular Biology 17(7):781-787; Hornung et al., (2006) Science 314(5801):994-997; Pichlmair et al., (2006) Science 314(5801):997-1001; Schlee et al., (2009) Immunity 31(1):25-34).). The crystal structures of RIG-FRNA complexes show protein binding to the backbone, not the bases, suggesting that the RNA sequence may not affect RIG-I binding or that RNA sequence may exhibit as of yet uncharacterized effects or activity. To date, evidence for sequence-dependent differential interaction or affinity with, and activation of, RIG-I-like receptors is not described in the art (Schlee and Hartmann (2010) Molecular Therapy 18(7): 1254-1262).

Accordingly, the disclosure provides synthetic RIG-I-like receptor (RLR) agonists comprising non-naturally occurring, synthetic, and or engineered RLR RNA ligands. In some aspects, the disclosure provides an RLR agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, and wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is selected from the group consisting of:

(i) a GT-repeat motif;

(ii) a GA-repeat motif;

(iii) a AUCG-repeat motif;

(iv) an AU-repeat motif;

(v) a dipyrimidine motif;

(vi) a dipurine motif;

(vii) a pyrimidine triplet motif;

(viii) a purine triplet motif;

(ix) a palindromic sequence motif; and

(x) a combination of any of (i)-(ix).

In some embodiments, the RLR agonists of the disclosure comprise at least one improved biological activity, wherein the improved biological activity is selected from:

(i) an increase in RLR-mediated cytokine production;

(ii) an increase in RLR-mediated expression of interferon-stimulated genes;

(iii) an increase in RLR-mediated intracellular signaling;

(iv) an increase in binding affinity to RLRs; and

(v) a combination of any of (i)-(iv).

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a GT-repeat motif comprises a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4 about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and thymine nucleotides, or derivatives or analogs thereof. In some embodiments, the GT-repeat motif is [GT]_(n), wherein n=2 to 9. In some embodiments, the GT-repeat motif is [GT]₇. In some embodiments, the GT-repeat motif is [GT]₃, and wherein the GT-repeat motif is followed by a purine triplet and UCG, respectively. In some embodiments, the purine triplet is GGA.

In some embodiments, the sequence motif is a GA-repeat motif comprises a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 5-10, about 5, about 4 about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 guanine and adenine nucleotides, or derivatives or analogs thereof. In some embodiments, the GA-repeat motif is [GA]_(n), where n=2 to 9. In some embodiments, the GA-repeat motif is [GA]₇.

In some embodiments, the RLR agonists of the disclosure comprise a sequence motif, wherein the sequence motif is a AUCG-repeat motif comprising a sequence of <19, about 16, about 12-16, about 12, about 8-12, about 6, about 16, 12, 8 adenine, uracil, cytosine, and guanine nucleotides, or derivatives or analogs thereof.

In some embodiments, the AUCG-repeat motif is [AUCG],, where n=2 to 4. In some embodiments, the AUCG-repeat motif is [AUCG]₃.

In some embodiments, the AUCG-repeat motif is preceded by a CG or a dipyrimidine motif. In some embodiments, the AUCG-repeat motif is preceded by a CG. In some embodiments, the dipyrimidine motif is CC. In some embodiments, the AUCG-repeat motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GA. In some embodiments, the dipurine motif is GG.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein one or more uridine nucleosides (U) are substituted with a modified nucleoside. In some embodiments, wherein the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC-repeat motif is [AUCG]₃, wherein the one or more uridine nucleosides (U) comprising the AUCG-repeat motif are substituted with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T). In some embodiments, the AUGC-repeat motif is [AUCG]₃, wherein the one or more uridine nucleosides (U) comprising the AUCG-repeat motif are substituted with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T), and wherein the AUGC-repeat motif is preceded by GG.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat motif, wherein one or more guanosine nucleosides (G) are substituted with a modified nucleoside. In some embodiments, the modified nucleoside is inosine (I). In some embodiments, the AUGC-repeat motif is [AUCG]₃, wherein the one or more guanosine nucleosides (G) comprising the AUCG-repeat motif are substituted with a modified nucleoside, wherein the modified nucleoside is ribothymidine (T), and wherein the AUGC-repeat motif is preceded by GG.

In some embodiments, the RLR agonists of the disclosure comprise a AUCG-repeat motif, wherein the motif is preceded by a IG. In some embodiments, the AUCG-repeat motif is [AUCG]₃ and is preceded by a IG.

In some embodiments, the RLR agonists of the disclosure comprise an AUCG-repeat, wherein one or more guanosine nucleosides (G) are substituted with an inosine (I), wherein the AUCG-repeat is preceded by an inosine (I). In some embodiments, the guanosine nucleosides (G) comprising the AUCG-repeat are substituted with an inosine (I), wherein the AUCG-repeat is preceded by an inosine (I), wherein the 5′ most nucleotide of the first polynucleotide comprises inosine (I).

In some embodiments, the 5′ most nucleotide of the first oligonucleotide comprises inosine (I).

In some embodiments, the RLR agonists of the disclosure comprise a AUCG-repeat sequence motif, wherein the AUCG-repeat motif is [AUCG]₂. In some embodiments, the AUCG-repeat motif is preceded by a dipurine motif. In some embodiments, the dipurine motif is GG. In some embodiments, the AUCG-repeat motif is preceded by a purine triplet. In some embodiments, the purine triplet is GGG. In some embodiments, the AUCG-repeat motif is preceded by CCCCCG. In some embodiments, the AUCG-repeat motif is preceded by TCGUCG.

In some embodiments, the RLR agonists of the disclosure comprise a palindromic sequence, wherein the palindromic sequence comprises a sequence of <19, about 15-18, about 15, about 10-15, about 10, about 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides, or derivatives or analogs thereof, linked in any order that results in a palindrome.

In some embodiments, the linker is flanked by AU. In some embodiments, the linker is flanked by an AU-repeat motif, wherein the AU-repeat motif is [AU]_(n), where n=2 to 3. In some embodiments, the AU-repeat motif is [AU]₂.

In some aspects, the disclosure provides an RLR agonist that specifically binds to a RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, and wherein the agonist comprises the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X2;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is a linker that covalently links the first polynucleotide and the second polynucleotide.

In other aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a non-nucleotide linker, and wherein the agonist comprises the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is the non-nucleotide linker that covalently links the first polynucleotide and the second polynucleotide.

In some embodiments, inosine, if present in the RLR agonist, base pairs with cytidine.

In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker.

In some aspects, the disclosure provides an RLR agonist that specifically binds to RLRs, wherein the agonist comprises a blunt-ended, hairpin RNA comprising a nucleotide or non-nucleotide linker. RNA hairpins are among the most common RNA secondary structural elements, wherein the hybridized portion or “stem” of the hairpin are frequently capped by RNA tetraloops. RNA tetraloops are composed of characteristic four-loop nucleotides that form a compact and stable structure. While they can be formed by many different nucleotide sequences, UNCG (N=A, C, G, or U), GNRA (R=A or G), and CUUG tetraloops are found most often. Tetraloops usually help initiate RNA-folding processes and provide sites for tertiary contacts within or between RNAs and for protein binding, thereby facilitating the assembly of ribonucleoprotein particles. Further description of tetraloops can be found in Cheong, H., Kim, N. and Cheong, C. (2015). RNA Structure: Tetraloops. In eLS, John Wiley & Sons, Ltd (Ed.), which is incorporated herein by reference in its entirety.

Accordingly, in some embodiments, the RLR agonists of the disclosure comprise a nucleotide linker comprising a tetraloop. In some embodiments, the nucleotide sequence of the tetraloop is selected from the group consisting of:

-   -   (a) UNCG, wherein N=A, C, G, or U;     -   (b) GNRA, wherein N=A, C, G, or U, and wherein R=A or G;     -   (c) ANYA, wherein N=A, C, G, or U, and wherein Y=C or T;     -   (d) CUYG, wherein Y=C or T;     -   (e) UMAC, wherein M=A or C; and     -   (f) CUUG.

In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the sequence of the tetraloop is UUCG. In some embodiments, the sequence of the tetraloop is GAUC. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

In other aspects, the RLR agonists of the disclosure comprise a non-nucleotide linker. As described herein nucleic acid loops (e.g., tetraloops) are a common element found in nucleic acid secondary structure. Nucleotide loops arise in folded domains occurring in intrastrand duplexes. Synthetic nucleic acids designed to contain hairpin loops comprising non-nucleotide linking groups (e.g., non-nucleotide linkers) can replace several nucleotides bridging a folded duplex structure. Non-nucleotide groups have been used as linkers in non-folded structures as well. Such linking groups may be useful replacements of natural nucleotide linkers (e.g., tetraloops). For example, they can shorten the synthesis of nucleic acid with a desired secondary structure by several steps, since one relatively long non-nucleotide linking group replaces several individual nucleotides which may normally constitute a loop. Such non-natural loops or linkers (e.g., non-nucleotide linkers) can confer resistance to degradation by nucleases which would ordinarily act on a natural loop structure in biological contexts (e.g., in a cell or in the circulation of a subject upon administration). A non-nucleotide linking group also has the potential to provide a more stable folded structure than occurs with the nucleotide loops and or linkers. Further description of non-nucleotide linkers can be found in Rumney and Kool (1995) J Am Chem Soc 117:5635-5646, which is incorporated herein by reference in its entirety.

Accordingly, in some embodiments, the RLR agonists of the disclosure comprise a non-nucleotide linker selected from the group consisting of:

-   -   (a) an ethylene glycol linker; and     -   (b) an alkyl linker.

In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the non-nucleotide linker is a C9 alkyl linker.

In some embodiments, the RLR agonist comprises a 5′ diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5′ triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog thereof comprises a phosphate bioisostere is selected from: a phosphonate, a thiophosphonate, a phosphorothioate, a sulfate, a sulfonate, a sulfamate, a thiazolidinone, a carboxylate, a malonate, a boronic acid, a benzoxaborole, a boranophosphate, a squaramide.

In some embodiments, the agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkages or to the polynucleotide backbone.

In some aspects, the RLR agonist of the disclosure exhibits at least one or more of the following properties:

-   -   (a) specifically binds to one or more RLRs (e.g. RIG-I, MDA5         and/or LGP2);     -   (b) increases RLR-mediated cytokine production;     -   (c) increases RLR-mediated expression of interferon-stimulated         genes (ISGs);     -   (d) increases RLR-dependent intracellular signaling;     -   (e) increases stability of the duplex;     -   (f) increases binding affinity to RLRs;     -   (g) decreases off-target binding;     -   (h) increases biological half-life;     -   (i) increases biodistribution and bioavailability;     -   (j) increases and/or enhances uptake into cells and/or tissues;     -   (k) decreases immunogenicity; and     -   (l) a combination of any of (a)-(k).

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ and X₂ are each oligonucleotides comprising nucleosides selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(viii) X₁ is complementary to X₂;

(ix) X₁ and X₂ are each 12 nucleotides to 16 nucleotides in length and are the same length, and;

(x) L is a linker that operably links the first polynucleotide and the second polynucleotide,

wherein at least one of N1, N2, N3, and N4 is inosine and/or at least one of X1 and/or X2 comprises at least one inosine nucleoside, and wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA.

In some embodiments, N1 comprises inosine and N4 comprises cytidine. In some embodiments, N1 comprises cytidine and N4 comprises inosine. In some embodiments, N2 comprise inosine and N3 comprises cytidine. In some embodiments, N2 comprises cytidine and N3 comprises inosine. In some embodiments, N1 comprises guanosine. In some embodiments, N2 comprises guanosine. In some embodiments, N1 comprises cytidine. In some embodiments, N2 comprises cytidine. In some embodiments, N1 and N2 comprise guanosine and N₃ and N₄ comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise guanosine. In some embodiments, N1 and N2 comprise inosine and N₃ and N₄ comprise cytidine. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise inosine. In some embodiments, N1 comprises inosine and N4 comprises cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N2 comprises inosine and N3 comprises cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine N₃ and N₄ comprise cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise guanosine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise guanosine and N3 and N4 comprise cytidine, and X1 and X2 each comprise inosine and no guanosine nucleosides. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise guanosine, and X1 and X2 each comprise inosine and no guanosine nucleosides. In some embodiments, N1 and N2 comprise inosine and N3 and N4 comprise cytidine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine and N3 and N4 comprise cytidine, and X1 and X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise cytidine and N3 and N4 comprise inosine, and X1 and/or X2 each comprise at least one inosine. In some embodiments, N1 and N2 comprise inosine and N3 and N4 comprise cytidine, and X1 and X2 comprise inosine and no guanosine nucleosides. In some embodiments, N1 and N2 comprise cytidine and N₃ and N₄ comprise inosine, and X1 and X2 comprise inosine and no guanosine nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides and comprise 1, 2, 3 or 4 inosine nucleosides. In some embodiments, X1 and X2 are each 13 nucleotides and comprise 1, 2, 3, 4 or 5 inosine nucleosides. In some embodiments, X1 and X2 are each 14 nucleotides and comprise 1, 2, 3, 4, 5 or 6 inosine nucleosides. In some embodiments, X1 and X2 are each 15 nucleotides and comprise 1, 2, 3, 4, 5, 6, or 7 inosine nucleosides. In some embodiments, X1 and X2 are each 16 nucleotides and each comprise 1, 2, 3, 4, 5, 6, 7, or 8 inosine nucleosides. In some embodiments, X1 and X2 are each 12 nucleotides and comprise at least 10%, 20%, 30% or 40% inosine nucleosides.

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula:

5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein

(i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁;

(ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄;

(iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine;

(iv) N₁ base pairs with N₄;

(v) N₂ base pairs with N₃;

(vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof;

(vii) X₁ comprises a sequence motif [AUCN5]_(x), wherein N₅ is comprises guanosine or inosine, wherein x is an integer whose value indicates the number of sequence motifs, and wherein x=3 or 4;

(viii) X₂ comprises a sequence motif [CN₆AU]_(y), wherein N6 comprises guanosine or inosine, wherein y is an integer whose value indicates the number of sequence motifs, and wherein y=3 or 4;

(ix) L is a linker that operably links the first polynucleotide and the second polynucleotide,

optionally, wherein at least one of N1, N2, N3, and N4 is inosine, and wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA. In some embodiments, N5 comprises inosine and N6 comprises inosine. In some embodiments, N5 comprises guanosine and N6 comprises inosine. In some embodiments, N5 comprises inosine and N6 comprises guanosine. In some embodiments, N5 comprises guanosine (G) and N6 comprises guanosine (G). In some embodiments, x=3 and y=3. In some embodiments, x=4 and y=4. In some embodiments, N1 comprises inosine (I) and N4 comprises cytidine (C). In some embodiments, N2 comprises inosine (I) and N3 comprises cytidine (C). In some embodiments, N3 comprises inosine (I) and N2 comprises cytidine (C). In some embodiments, N4 comprises inosine (I) and N1 comprises cytidine (C). In some embodiments, N1 comprises guanosine (G). In some embodiments, N2 comprises guanosine (G). In some embodiments, N1 comprises cytidine (C). In some embodiments, N2 comprises cytidine (C). In some embodiments, N1 and N2 comprise guanosine (G) and N₃ and N₄ comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N₃ and N₄ comprise guanosine (G). In some embodiments, N1 and N2 comprise inosine (I) and N₃ and N₄ comprise cytidine (C). In some embodiments, N1 and N2 comprise cytidine (C) and N₃ and N₄ comprise inosine (I).

In some embodiments, the linker (L) is a nucleotide linker or a non-nucleotide linker. In some embodiments, the linker (L) is a nucleotide linker comprising a tetraloop, wherein the nucleotide sequence of the tetraloop is selected from the group consisting of:

-   -   (a) UNCG, wherein N=A, C, G, or U;     -   (b) GNRA, wherein N=A, C, G, or U, and wherein R=A or G;     -   (c) ANYA, wherein N=A, C, G, or U, and wherein Y=C or T;     -   (d) CUYG, wherein Y=C or T;     -   (e) UMAC, wherein M=A or C; and     -   (f) CUUG.

In some embodiments, the linker (L) is a nucleotide linker comprising the nucleotide sequence UUUGAU or UGUUU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UUUGAU. In some embodiments, the nucleotide linker comprises the nucleotide sequence UGUUU.

In some embodiments, the linker (L) is a nucleotide linker comprising a tetraloop, wherein the sequence of the tetraloop is UUCG. In some embodiments, the sequence of the tetraloop is GAUC.

In some embodiments, the linker (L) is a non-nucleotide linker selected from the group consisting of:

-   -   (a) an ethylene glycol linker; and     -   (b) an alkyl linker.

In some embodiments, the non-nucleotide linker is a hexaethylene glycol linker. In some embodiments, the the non-nucleotide linker is a C9 alkyl linker.

In some embodiments, the RLR agonist comprises a 5′ diphosphate moiety, or a derivative or analog thereof. In some embodiments, the agonist comprises a 5′ triphosphate moiety, or a derivative or analog thereof. In some embodiments, the derivative or analog thereof comprises a phosphate bioisostere is selected from: a phosphonate, a thiophosphonate, a phosphorothioate, a sulfate, a sulfonate, a sulfamate, a thiazolidinone, a carboxylate, a malonate, a boronic acid, a benzoxaborole, a boranophosphate, a squaramide.

In some embodiments, the RLR agonist comprises a modified nucleotide, a modified nucleoside, or a modified nucleobase, or a combination thereof. In some embodiments, the agonist comprises a modification to the internucleotide linkages or to the polynucleotide backbone.

In some embodiments, the RLR agonist exhibits at least one or more of the following properties:

-   -   (a) specifically binds to one or more RLRs (e.g. RIG-1, MDA5         and/or LGP2);     -   (b) increases RLR-mediated cytokine production;     -   (c) increases RLR-mediated expression of interferon-stimulated         genes (ISGs);     -   (d) increases RLR-dependent intracellular signaling;     -   (e) increases stability of the duplex;     -   (f) increases binding affinity to RLRs;     -   (g) decreases off-target binding;     -   (h) increases biological half-life;     -   (i) increases biodistribution and bioavailability;     -   (j) increases and/or enhances uptake into cells and/or tissues;     -   (k) decreases immunogenicity; and     -   (l) a combination of any of (a)-(k).

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, and wherein the agonist comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof, wherein the agonist comprises a sequence motif that provides at least one improved biological activity mediated by the RLR relative to an agonist that does not comprise the sequence motif, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of:

(i) SEQ ID NO: 37 and 68, respectively;

(ii) SEQ ID NO: 38 and 69, respectively;

(iii) SEQ ID NO: 39 and 70, respectively;

(iv) SEQ ID NO: 40 and 71, respectively;

(v) SEQ ID NO: 41 and 72, respectively;

(vi) SEQ ID NO: 42 and 73, respectively;

(vii) SEQ ID NO: 43 and 74, respectively;

(viii) SEQ ID NO: 44 and 75, respectively;

(ix) SEQ ID NO: 45 and 76, respectively;

(x) SEQ ID NO: 46 and 77, respectively;

(xi) SEQ ID NO: 47 and 78, respectively;

(xii) SEQ ID NO: 48 and 79, respectively;

(xiii) SEQ ID NO: 49 and 80, respectively;

(xiv) SEQ ID NO: 50 and 81, respectively;

(xv) SEQ ID NO: 51 and 82, respectively;

(xvi) SEQ ID NO: 52 and 83, respectively;

(xvii) SEQ ID NO: 53 and 84, respectively;

(xviii) SEQ ID NO: 54 and 85, respectively;

(xix) SEQ ID NO: 55 and 86, respectively;

(xx) SEQ ID NO: 56 and 87, respectively;

(xxi) SEQ ID NO: 57 and 88, respectively;

(xxii) SEQ ID NO: 58 and 89, respectively;

(xxiii) SEQ ID NO: 59 and 89, respectively;

(xxiv) SEQ ID NO: 60 and 90, respectively;

(xxv) SEQ ID NO: 61 and 91, respectively;

(xxvi) SEQ ID NO: 62 and 92, respectively;

(xxvii) SEQ ID NO: 63 and 91, respectively;

(xxviii) SEQ ID NO: 64 and 93, respectively;

(xxix) SEQ ID NO: 65 and 94, respectively;

(xxx) SEQ ID NO: 66 and 95, respectively;

(xxxi) SEQ ID NO: 67 and 96, respectively; and

(xxxii) SEQ ID NO: 63 and 97, respectively.

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, and wherein the agonist comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23 and 25.

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, wherein the agonist comprises the formula 5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein (N₁-N₂-X₁) comprises a first polynucleotide and (X2-N₃-N₄) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of:

-   -   (i) SEQ ID NO: 58 and 89, respectively;     -   (ii) SEQ ID NO: 59 and 89, respectively; and     -   (iii) SEQ ID NO: 61 and 91, respectively.

In some aspects, the disclosure provides a synthetic RIG-I-like receptor (RLR) agonist that specifically binds to RIG-I-like receptors (RLRs), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a non-nucleotide linker, wherein the agonist comprises the formula 5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein (N₁-N₂-X₁) comprises a first polynucleotide and (X₂-N₃-N₄) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of:

(i) SEQ ID NO: 37 and 68, respectively;

(ii) SEQ ID NO: 38 and 69, respectively;

(iii) SEQ ID NO: 39 and 70, respectively;

(iv) SEQ ID NO: 40 and 71, respectively;

(v) SEQ ID NO: 41 and 72, respectively;

(vi) SEQ ID NO: 42 and 73, respectively;

(vii) SEQ ID NO: 43 and 74, respectively;

(viii) SEQ ID NO: 44 and 75, respectively;

(ix) SEQ ID NO: 45 and 76, respectively;

(x) SEQ ID NO: 46 and 77, respectively;

(xi) SEQ ID NO: 47 and 78, respectively;

(xii) SEQ ID NO: 48 and 79, respectively;

(xiii) SEQ ID NO: 49 and 80, respectively;

(xiv) SEQ ID NO: 50 and 81, respectively;

(xv) SEQ ID NO: 51 and 82, respectively;

(xvi) SEQ ID NO: 52 and 83, respectively;

(xvii) SEQ ID NO: 53 and 84, respectively;

(xviii) SEQ ID NO: 54 and 85, respectively;

(xix) SEQ ID NO: 55 and 86, respectively;

(xx) SEQ ID NO: 56 and 87, respectively;

(xxi) SEQ ID NO: 57 and 88, respectively;

(xxii) SEQ ID NO: 58 and 89, respectively;

(xxiii) SEQ ID NO: 59 and 89, respectively;

(xxiv) SEQ ID NO: 60 and 90, respectively;

(xxv) SEQ ID NO: 61 and 91, respectively;

(xxvi) SEQ ID NO: 62 and 92, respectively;

(xxvii) SEQ ID NO: 63 and 91, respectively;

(xxviii) SEQ ID NO: 64 and 93, respectively;

(xxix) SEQ ID NO: 65 and 94, respectively;

(xxx) SEQ ID NO: 66 and 95, respectively;

(xxxi) SEQ ID NO: 67 and 96, respectively; and

(xxxii) SEQ ID NO: 63 and 97, respectively.

In some aspects, the disclosure provides RLR agonists wherein the nucleotide sequence comprising the agonist is not complementary to a genomic DNA sequence or mRNA sequence, wherein the RLR agonist does not participate in RNA interference, and wherein the RLR agonist does not silence gene expression.

RLR Agonists Comprising Modified Nucleobases, Nucleosides, or Nucleotides

In some embodiments, an RLR agonist of the disclosure comprises one or more modified nucleobases, nucleosides, or nucleotides. In some embodiments, modified RLR agonists may have useful properties, including enhanced stability, intracellular retention, enhanced target binding, and/or an increase in induction of the innate immune response of a cell into which the RLR agonist is introduced, as compared to a reference unmodified RLR agonist. Therefore, use of modified RLR agonists may enhance the efficiency of target binding, intracellular retention of nucleic acids, as well as possess reduced immunogenicity. In one embodiment, the agonist provided by the disclosure is comprised of one or more oligonucleotides that comprise at least one region modified to increase target binding affinity. Affinity of an oligonucleotide for its target polypeptide (e.g. an RLR receptor) can be determined by, for example, measuring the degree of fluorescence polarization (FP) upon binding of a fluorescently-labeled oligonucleotide to its target (Moerke (2009) Curr Protoc Chem Biol 1(1):1-15).

In another embodiment, the RLR agonist provided by the disclosure is comprised of at least one oligonucleotide comprising at least one region comprising at least one modified nucleobase, nucleoside, or nucleotide that increases the stability of the duplex. The stability of the duplex can be routinely determined by measuring the Tm of the duplex, which is the temperature at which the two oligonucleotide strands comprising the duplex dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the stability of the duplex.

In one embodiment, the region of the oligonucleotide which is modified to increase duplex stability comprises at least one nucleotide modified at the 2′ position of the sugar, most preferably a 2′-O-alkyl, 2-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. In another embodiment, an oligonucleotide comprising an RLR agonist is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than an unmodified oligonucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance (De Mesmaeker et al., 1995, Acc. Chem. Res. 28:366-374).

Specific examples of some preferred oligonucleotides envisioned for this invention include those containing modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones (including those synthesized in a stereo-specific manner) and those with heteroatom backbones, particularly CH2-NH—O—CH2, CH2-N(CH3)-O—CH2 [known as a methylene(methylimino) or MMI backbone], CH2-O—N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and O—N(CH3)-CH2-CH2 backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH2). The amide backbones disclosed by De Mesmaeker et al. (1995, Acc. Chem. Res. 28:366-374) are also preferred. Oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; C1to C10 lower alkyl, alkoxyalkoxy (also known in the art as O-alkyl-O-alkyl), substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O, S—, or N-alkyl; O—O, S—, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-propoxy (2′-OCH2CH2CH3) and 2′-fluoro (2′-F) . Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′deoxycytosine and often referred to in the art as 5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp75-77; Gebeyehu, G., et al., 1987, Nucl. Acids Res. 15:4513). A “universal” base known in the art, e.g., inosine, may be included. 5-me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651). Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255.

The oligonucleotides of the invention may be provided as prodrugs, which comprise one or more moieties which are cleaved off, generally in the body, to yield an active oligonucleotide. One example of a prodrug approach is described by Imbach et al. in WO Publication 94/26764.

It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

The oligonucleotides in accordance with this invention preferably are from about 8 to about 50 nucleotides in length. In the context of this invention it is understood that this encompasses non-naturally occurring oligomers as hereinbefore described, having 8 to 50 monomers.

The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the knowledge and ability of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling Va.) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.

In some embodiments, an RLR agonist includes one or more (e.g., 1, 2, 3 or 4) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, an RLR agonist includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more) different modified nucleobases, nucleosides, or nucleotides. In some embodiments, the modified RLR agonist may have reduced degradation in a cell into which the RLR agonist is introduced, relative to a corresponding unmodified RLR agonist.

In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ S²U), 5-aminomethyl-2-thio-uridine (nm ⁵ S²U), 5-methyl aminomethyl-uridine (mnm⁵U), 5-methyl aminomethyl-2-thio-uridine (mnm ⁵ S²U), 5-methyl aminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethyl aminomethyl-2-thio-uridine (cmnm ⁵ s²U), 5-propynyl -uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm ⁵ S²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ), 5-methyl-2-thio-uridine (m⁵ s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl -1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵ s²U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2 ′-O-methyl-uridine (s²Um), 5-methoxycarbonylmethyl-2 ′-O-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2 ′-O-methyl-uridine (cmnm⁵Um), 3,2 ′-O-dimethyl-uridine (m³Um), and 5-(isopentenylaminomethyl)-2¹-O-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2¹-O-dimethyl-cytidine (m⁵ Cm), N4-acetyl-2¹-O-methyl-cytidine (ac⁴Cm), N4,2-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2¹-O-methyl-cytidine (f⁵Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include a-thio-adenosine, 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A), 2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms²m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis-hydroxyisopentenyl)adenosine (io⁶A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A), N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶ ₂Am), 1,2¹-O-dimethyl-adenosine (m¹Am), 2¹-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include α-thio-guanosine, inosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀), 7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G), N2,N2-dimethyl-guanosine (m²2G), N2,7-dimethyl-guanosine (m^(2,7)G), N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm), 1-methyl-2′-O-methyl-guanosine (m¹Gm), N2,7-dimethyl-2¹-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2′-F-ara-guanosine, and 2′-F-guanosine.

In some embodiments, an RLR agonist of the disclosure includes a combination of one or more of the aforementioned modified nucleobases (e.g., a combination of 2, 3 or 4 of the aforementioned modified nucleobases.)

In certain embodiments, an RLR agonist of the disclosure is uniformly modified (i.e., fully modified, modified through-out the entire sequence) for a particular modification. For example, an RLR agonist can be uniformly modified with 5-methyl-cytidine (m⁵C), meaning that all cytosine residues in the mRNA sequence are replaced with 5-methyl-cytidine (m⁵C). Similarly, an RLR agonist of the disclosure can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

Examples of nucleoside modifications and combinations thereof that may be present in an RLR agonist of the present disclosure include, but are not limited to, those described in PCT Patent Application Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, and WO2014159813.

The RLR agonists of the disclosure can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein.

Examples of modified nucleosides and modified nucleoside combinations are provided below in Table 1 and Table 2. These combinations of modified nucleotides can be used to form the RLR agonists of the disclosure. In certain embodiments, the modified nucleosides may be partially or completely substituted for the natural nucleotides of the RLR agonists of the disclosure. As a non-limiting example, the natural nucleotide uridine may be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleoside uridine may be partially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9% of the natural uridines) with at least one of the modified nucleoside disclosed herein.

TABLE 1 Combinations of Nucleoside Modifications Modified Nucleotide Modified Nucleotide Combination α-thio-cytidine α-thio-cytidine/5-iodo-uridine α-thio-cytidine/N1-methyl-pseudouridine α-thio-cytidine/α-thio-uridine α-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about 50% of the cytosines are α-thio-cytidine pseudoisocytidine pseudoisocytidine/5-iodo-uridine pseudoisocytidine/N1-methyl-pseudouridine pseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridine pseudoisocytidine/pseudouridine about 25% of cytosines are pseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudouridine pseudoisocytidine/about 25% of uridines are N1-methyl-pseudouridine and about 25% of uridines are pseudouridine pyrrolo-cytidine pyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridine pyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridine pyrrolo-cytidine/pseudouridine about 50% of the cytosines are pyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine 5-methyl-cytidine/N1-methyl-pseudouridine 5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine 5-methyl-cytidine/pseudouridine about 25% of cytosines are 5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine 5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine 5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridines are 2- thio-uridine about 50% of uridines are 5-methyl-cytidine/ about 50% of uridines are 2-thio-uridine N4-acetyl-cytidine N4-acetyl-cytidine/5-iodo-uridine N4-acetyl-cytidine/N1-methyl-pseudouridine N4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridine N4-acetyl-cytidine/pseudouridine about 50% of cytosines are N4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidine N4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridine N4-acetyl-cytidine/2-thio-uridine about 50% of cytosines are N4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine

TABLE 2 Modified Nucleosides and Combinations Thereof 1-(2,2,2-Trifluoroethyl)pseudo-UTP 1-Ethyl-pseudo-UTP 1-Methyl-pseudo-U-alpha-thio-TP 1-methyl-pseudouridine TP, ATP, GTP, CTP 1-methyl-pseudo-UTP/5-methyl-CTP/ATP/GTP 1-methyl-pseudo-UTP/CTP/ATP/GTP 1-Propyl-pseudo-UTP 25% 5-Aminoallyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Aminoallyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Bromo-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP + 75% CTP/1-Methyl-pseudo-UTP 25% 5-Carboxy-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Carboxy-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Ethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Ethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Ethynyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Ethynyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Fluoro-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Fluoro-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Formyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Formyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Hydroxymethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Iodo-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Iodo-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Methoxy-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Methoxy-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/50% 5-Methoxy-UTP + 50% UTP 25% 5-Methyl-CTP + 75% CTP/5-Methoxy-UTP 25% 5-Methyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 25% 5-Methyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Phenyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Phenyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Trifluoromethyl-CTP + 75% CTP/1-Methyl-pseudo-UTP 25% N4-Ac-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Ac-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% N4-Bz-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Bz-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% N4-Methyl-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% N4-Methyl-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% Pseudo-iso-CTP + 75% CTP/25% 5-Methoxy-UTP + 75% UTP 25% Pseudo-iso-CTP + 75% CTP/75% 5-Methoxy-UTP + 25% UTP 25% 5-Bromo-CTP/75% CTP/Pseudo-UTP 25% 5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 25% 5-methoxy-UTP/CTP/ATP/GTP 25% 5-metoxy-UTP/50% 5-methyl-CTP/ATP/GTP 2-Amino-ATP 2-Thio-CTP 2-thio-pseudouridine TP, ATP, GTP, CTP 2-Thio-pseudo-UTP 2-Thio-UTP 3-Methyl-CTP 3-Methyl-pseudo-UTP 4-Thio-UTP 50% 5-Bromo-CTP + 50% CTP/1-Methyl-pseudo-UTP 50% 5-Hydroxymethyl-CTP + 50% CTP/1-Methyl-pseudo-UTP 50% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 50% 5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 50% 5-Methyl-CTP + 50% CTP/25% 5-Methoxy-UTP + 75% UTP 50% 5-Methyl-CTP + 50% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP 50% 5-Methyl-CTP + 50% CTP/50% 5-Methoxy-UTP + 50% UTP 50% 5-Methyl-CTP + 50% CTP/5-Methoxy-UTP 50% 5-Methyl-CTP + 50% CTP/75% 5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 50% 5-Methyl-CTP + 50% CTP/75% 5-Methoxy-UTP + 25% UTP 50% 5-Trifluoromethyl-CTP + 50% CTP/1-Methyl-pseudo-UTP 50% 5-Bromo-CTP/50% CTP/Pseudo-UTP 50% 5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/50% 5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 50% 5-methoxy-UTP/CTP/ATP/GTP 5-Aminoallyl-CTP 5-Aminoallyl-CTP/5-Methoxy-UTP 5-Aminoallyl-UTP 5-Bromo-CTP 5-Bromo-CTP/5-Methoxy-UTP 5-Bromo-CTP/1-Methyl-pseudo-UTP 5-Bromo-CTP/Pseudo-UTP 5-bromocytidine TP, ATP, GTP, UTP 5-Bromo-UTP 5-Carboxy-CTP/5-Methoxy-UTP 5-Ethyl-CTP/5-Methoxy-UTP 5-Ethynyl-CTP/5-Methoxy-UTP 5-Fluoro-CTP/5-Methoxy-UTP 5-Formyl-CTP/5-Methoxy-UTP 5-Hydroxy-methyl-CTP/5-Methoxy-UTP 5-Hydroxymethyl-CTP 5-Hydroxymethyl-CTP/1-Methyl-pseudo-UTP 5-Hydroxymethyl-CTP/5-Methoxy-UTP 5-hydroxymethyl-cytidine TP, ATP, GTP, UTP 5-Iodo-CTP/5-Methoxy-UTP 5-Me-CTP/5-Methoxy-UTP 5-Methoxy carbonyl methyl-UTP 5-Methoxy-CTP/5-Methoxy-UTP 5-methoxy-uridine TP, ATP, GTP, UTP 5-methoxy-UTP 5-Methoxy-UTP 5-Methoxy-UTP/N6-Isopentenyl-ATP 5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 5-methoxy-UTP/CTP/ATP/GTP 5-Methyl-2-thio-UTP 5-Methylaminomethyl-UTP 5-Methyl-CTP/5-Methoxy-UTP 5-Methyl-CTP/5-Methoxy-UTP(cap 0) 5-Methyl-CTP/5-Methoxy-UTP(No cap) 5-Methyl-CTP/25% 5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP 5-Methyl-CTP/25% 5-Methoxy-UTP + 75% UTP 5-Methyl-CTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP 5-Methyl-CTP/50% 5-Methoxy-UTP + 50% UTP 5-Methyl-CTP/5-Methoxy-UTP/N6-Me-ATP 5-Methyl-CTP/75% 5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP 5-Methyl-CTP/75% 5-Methoxy-UTP + 25% UTP 5-Phenyl-CTP/5-Methoxy-UTP 5-Trifluoro-methyl-CTP/5-Methoxy-UTP 5-Trifluoromethyl-CTP 5-Trifluoromethyl-CTP/5-Methoxy-UTP 5-Trifluoromethyl-CTP/1-Methyl-pseudo-UTP 5-Trifluoromethyl-CTP/Pseudo-UTP 5-Trifluoromethyl-UTP 5-trifluromethylcytidine TP, ATP, GTP, UTP 75% 5-Aminoallyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Aminoallyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Bromo-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Carboxy-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Carboxy-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Ethyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Ethyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Ethynyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Ethynyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Fluoro-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Fluoro-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Formyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Formyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Hydroxymethyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Hydroxymethyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Iodo-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Iodo-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Methoxy-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Methoxy-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-methoxy-UTP/5-methyl-CTP/ATP/GTP 75% 5-Methyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Methyl-CTP + 25% CTP/50% 5-Methoxy-UTP + 50% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25% CTP/50% 5-Methoxy-UTP + 50% UTP 75% 5-Methyl-CTP + 25% CTP/5-Methoxy-UTP 75% 5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% 1-Methyl- pseudo-UTP 75% 5-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Phenyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Phenyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Trifluoromethyl-CTP + 25% CTP/1-Methyl-pseudo-UTP 75% N4-Ac-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% N4-Ac-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% N4-Bz-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% N4-Bz-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% N4-Methyl-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% N4-Methyl-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% Pseudo-iso-CTP + 25% CTP/25% 5-Methoxy-UTP + 75% UTP 75% Pseudo-iso-CTP + 25% CTP/75% 5-Methoxy-UTP + 25% UTP 75% 5-Bromo-CTP/25% CTP/1-Methyl-pseudo-UTP 75% 5-Bromo-CTP/25% CTP/Pseudo-UTP 75% 5-methoxy-UTP/25% 5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/50% 5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/75% 5-methyl-CTP/ATP/GTP 75% 5-methoxy-UTP/CTP/ATP/GTP 8-Aza-ATP Alpha-thio-CTP CTP/25% 5-Methoxy-UTP + 75% 1-Methyl-pseudo-UTP CTP/25% 5-Methoxy-UTP + 75% UTP CTP/50% 5-Methoxy-UTP + 50% 1-Methyl-pseudo-UTP CTP/50% 5-Methoxy-UTP + 50% UTP CTP/5-Methoxy-UTP CTP/5-Methoxy-UTP (cap 0) CTP/5-Methoxy-UTP(No cap) CTP/75% 5-Methoxy-UTP + 25% 1-Methyl-pseudo-UTP CTP/75% 5-Methoxy-UTP + 25% UTP CTP/UTP(No cap) N1-Me-GTP N4-Ac-CTP N4Ac-CTP/1-Methyl-pseudo-UTP N4Ac-CTP/5-Methoxy-UTP N4-acetyl-cytidine TP, ATP, GTP, UTP N4-Bz-CTP/5-Methoxy-UTP N4-methyl CTP N4-Methyl-CTP/5-Methoxy-UTP Pseudo-iso-CTP/5-Methoxy-UTP PseudoU-alpha-thio-TP pseudouridine TP, ATP, GTP, CTP pseudo-UTP/5-methyl-CTP/ATP/GTP UTP-5-oxyacetic acid Me ester Xanthosine

According to the disclosure, polynucleotides of the disclosure may be synthesized to comprise the combinations or single modifications of Table 1 or Table 2.

Where a single modification is listed, the listed nucleoside or nucleotide represents 100 percent of that A, U, G or C nucleotide or nucleoside having been modified. Where percentages are listed, these represent the percentage of that particular A, U, G or C nucleobase triphosphate of the total amount of A, U, G, or C triphosphate present. For example, the combination: 25% 5-Aminoallyl-CTP+75% CTP/25% 5-Methoxy-UTP+75% UTP refers to a polynucleotide where 25% of the cytosine triphosphates are 5-Aminoallyl-CTP while 75% of the cytosines are CTP; whereas 25% of the uracils are 5-methoxy UTP while 75% of the uracils are UTP. Where no modified UTP is listed then the naturally occurring ATP, UTP, GTP and/or CTP is used at 100% of the sites of those nucleotides found in the polynucleotide. In this example all of the GTP and ATP nucleotides are left unmodified.

Methods of Making RLR Agonists

RLR agonists of the present disclosure may be produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic (IVT), solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In one embodiment, RLR agonists are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT/US2013/30062, the contents of which are incorporated herein by reference in their entirety. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an RLR agonist described herein.

Non-natural modified nucleobases may be introduced into polynucleotides, e.g., RNA, during synthesis or post-synthesis. In certain embodiments, modifications may be on internucleoside linkages, purine or pyrimidine bases, or sugar. In particular embodiments, the modification may be introduced at the terminal of a polynucleotide chain or anywhere else in the polynucleotide chain; with chemical synthesis or with a polymerase enzyme. Examples of modified nucleic acids and their synthesis are disclosed in PCT application No. PCT/US2012/058519. Synthesis of modified polynucleotides is also described in Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998).

Either enzymatic or chemical ligation methods may be used to conjugate polynucleotides or their regions with different functional moieties, such as targeting or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990). The synthesis of oligonucleotides, polynucleotides, and conjugations and ligations thereof, is further described in Taskova et al., (2017) Chembiochem 18(17):1671-1682; Gooding et al., (2016) Eur J Pharm Biopharm 107:321-40; Menzi et al., (2015) Future Med Chem 7(13):1733-49; Winkler J., (2013) Ther Deliv. (7):791-809; Singh et al., (2010) Chem Soc Rev 39(6):2054-70; and Lu et al., (2010) Bioconjug Chem 21(2):187-202.

Pharmaceutical Compositions and Formulations

In certain embodiments, the invention provides for a pharmaceutical composition comprising an RLR agonist with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for s.c. and/or I.V. administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In certain embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of the RLR agonist.

In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In certain embodiments, a composition comprising an RLR agonist can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising an RLR agonist can be formulated as a lyophilizate using appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an RLR agonist, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which an RLR agonist is formulated as a sterile, isotonic solution, and properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with a delivery vehicle or agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid, polyglycolic acid or polyethylenimine (e.g. JetPEI®)), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, an RLR agonist can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising an RLR agonist can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in PCT application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, an RLR agonist that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of an RLR agonist. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve an effective quantity of an RLR agonist in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving an RLR agonist in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this can be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.

In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceutical composition comprising an RLR agonist to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which an RLR agonist is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.

In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of an RLR agonist in the formulation used. In certain embodiments, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, individual elements of the combination therapy may be administered by different routes.

In certain embodiments, the composition can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. In certain embodiments, it can be desirable to use a pharmaceutical composition comprising an RLR agonist in an ex vivo manner. In such instances, cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising an RLR agonist after which the cells, tissues and/or organs are subsequently implanted back into the patient.

In certain embodiments, an RLR agonist can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the agonist. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.

In some aspects, the disclosure provides a pharmaceutical composition comprising an RLR agonist according to the disclosure for stimulating an immune response, treating or delaying progression of a cancer, or reducing or inhibiting tumor growth in a subject in need thereof, and a pharmaceutically acceptable carrier. In some embodiments, the RLR agonist is formulated in a polyethylenimine (PEI) carrier. In some embodiments, the PEI carrier is JetPEI®.

Applications

The compositions described herein can be used in diagnostic and therapeutic applications. For example, detectably-labeled RLR agonists can be used in assays to detect the presence or amount of the target protein in a sample (e.g., a biological sample). The compositions can be used in in vitro assays for studying inhibition of target function (e.g. RLR-mediated cellular signaling or response). In some embodiments, e.g., in which the compositions bind to and activate a target (e.g. a protein or polypeptide), the compositions can be used as positive controls in assays designed to identify additional novel compounds that also induce activity of the target protein or polypeptide and/or are otherwise are useful for treating a disorder associated with the target protein or polypeptide. For example, a RLR-activating composition can be used as a positive control in an assay to identify additional compounds (e.g., small molecules, aptamers, or antibodies) that induce, increase, or stimulate RLR function. The compositions can also be used in therapeutic methods as elaborated on below.

Kits

A kit can include an RLR agonist as disclosed herein, and instructions for use. The kits may comprise, in a suitable container, an RLR agonist, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art.

The container can include at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which an RLR agonist may be placed, and in some instances, suitably aliquoted. Where an additional component is provided, the kit can contain additional containers into which this component may be placed. The kits can also include a means for containing an RLR agonist and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and/or kits can include labeling with instructions for use and/or warnings.

In some aspects, the disclosure provides a kit comprising an RLR agonist provided by the disclosure, or comprising a pharmaceutical composition provided by the disclosure and instructions for use in stimulating an immune response in a subject, or treating or delaying progression of a cancer, or inhibiting tumor growth in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents.

In some embodiments, the agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cell death-inducing agent, an opsonizing agent (e.g., an opsonizing antibody) a cytotoxic agent, an immune-based therapy, a cytokine, an activator of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, a cellular immunotherapy, or a combination thereof.

In some embodiments, the RLR agonist or pharmaceutical composition is administered preceding or subsequent to administration of the one or more additional therapeutic agents or wherein the one or more additional therapeutic agents is administered concurrently with, preceding or subsequent to the administration of the RLR agonist or pharmaceutical composition.

In some embodiments, the one or more additional therapeutic agents is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a toll-like receptor 3 (TLR3) agonist, a toll-like receptor 7 (TLR7) agonist, a toll-like receptor 9 (TLR9) agonist.

In some embodiments, the one or more additional therapeutic agents is an agonist comprising an polypeptide (e.g, antibody, or antigen binding portion thereof) that specifically binds to CD137 (4-1BB).

In some embodiments, the one or more additional therapeutic agents is an agonist comprising an polypeptide (e.g., antibody, or antigen binding portion thereof) that specifically binds to CD134 (OX40).

Methods of Use

The compositions of the present invention have numerous in vitro and in vivo utilities involving the detection and/or quantification of RLRs and/or the agonism of RLR function.

The above-described compositions are useful in, inter alia, methods for treating or preventing a variety of cancers or infectious diseases in a subject. The compositions can be administered to a subject, e.g., a human subject, using a variety of methods that depend, in part, on the route of administration. The route can be, e.g., intravenous injection or infusion (IV), subcutaneous injection (SC), intradermal injection (ID), intraperitoneal (IP) injection, intramuscular injection (IM), intratumoral injection (IT) or intrathecal injection. The injection can be in a bolus or a continuous infusion.

Administration can be achieved by, e.g., local infusion, injection, or by means of an implant. The implant can be of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. The implant can be configured for sustained or periodic release of the composition to the subject. See, e.g., U.S. Patent Application Publication No. 20080241223; U.S. Pat. Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety. The composition can be delivered to the subject by way of an implantable device based on, e.g., diffusive, erodible, or convective systems, e.g., osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmosis systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

In some embodiments, an RLR agonist is therapeutically delivered to a subject by way of local administration.

A suitable dose of an RLR agonist described herein, which dose is capable of treating or preventing cancer in a subject, can depend on a variety of factors including, e.g., the age, sex, and weight of a subject to be treated and the particular inhibitor compound used. Other factors affecting the dose administered to the subject include, e.g., the type or severity of the cancer or infectious disease. For example, a subject having metastatic melanoma may require administration of a different dosage of an RLR agonist than a subject with glioblastoma. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the general health of the subject, the genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject will also depend upon the judgment of the treating medical practitioner (e.g., doctor or nurse). Suitable dosages are described herein.

A pharmaceutical composition can include a therapeutically effective amount of an RLR agonist thereof described herein. Such effective amounts can be readily determined by one of ordinary skill in the art based, in part, on the effect of the administered RLR agonist, or the combinatorial effect of the RLR agonist and one or more additional active agents, if more than one agent is used. A therapeutically effective amount of an RLR agonist described herein can also vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agonist (and one or more additional active agents) to elicit a desired response in the individual, e.g., reduction in tumor growth. For example, a therapeutically effective amount of an RLR agonist can inhibit (lessen the severity of or eliminate the occurrence of) and/or prevent a particular disorder, and/or any one of the symptoms of the particular disorder known in the art or described herein. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

Suitable human doses of any of the RLR agonists described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8 (8): 1711-1718 ; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1) : 523-531 ; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500.

In some embodiments, the composition contains any of the RLR agonists described herein and one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, or 11 or more) additional therapeutic agents such that the composition as a whole is therapeutically effective. For example, a composition can contain an RLR agonist described herein and an alkylating agent, wherein the agonist and agent are each at a concentration that when combined are therapeutically effective for treating or preventing a cancer (e.g., melanoma) in a subject.

Toxicity and therapeutic efficacy of such compositions can be determined by known pharmaceutical procedures in cell cultures or experimental animals (e.g., animal models of any of the cancers described herein). These procedures can be used, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. An RLR agonist that exhibits a high therapeutic index is preferred. While compositions that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue and to minimize potential damage to normal cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. For an RLR agonist described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC₅₀ (i.e., the concentration of the agonist which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. In some embodiments, e.g., where local administration (e.g., to the eye or a joint) is desired, cell culture or animal modeling can be used to determine a dose required to achieve a therapeutically effective concentration within the local site.

In some embodiments, the methods can be performed in conjunction with other therapies for cancer or infectious disease. For example, the composition can be administered to a subject at the same time, prior to, or after, radiation, surgery, targeted or cytotoxic chemotherapy, chemoradiotherapy, hormone therapy, immunotherapy, gene therapy, cell transplant therapy, precision medicine, genome editing therapy, or other pharmacotherapy.

As described above, the compositions described herein (e.g., RLR agonist compositions) can be used to treat a variety of cancers such as but not limited to: Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma, marginal zone B cell lymphoma, polycythemia vera, Hodgkin's disease, non-Hodgkin' s disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cavity cancer (for example lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, rectal cancer; cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system.

In some aspects, the disclosure provides a method to increase RLR-mediated production of one or more cytokines in a cell, the method comprising contacting the cell with an RLR agonist provided by the disclosure, wherein the agonist increases RLR-mediated cytokine production in a cell.

In some aspects, the disclosure provides a method to increase RLR-mediated expression of one or more interferon-stimulated genes in a cell, the method comprising contacting the cell with an RLR agonist provided by the disclosure, wherein the agonist increases RLR-mediated expression of one or more interferon-stimulated genes in a cell.

In some aspects, the disclosure provides a method to increase RLR-dependent intracellular signaling in a cell, the method comprising contacting the cell with an RLR agonist provided by the disclosure, wherein the agonist increases RLR-dependent intracellular signaling.

In some aspects, the disclosure provides a method of stimulating an immune response in a subject, the method comprising administering to the subject an effective amount of an RLR agonist provided by the disclosure, or a pharmaceutical composition provided by the disclosure.

In some aspects, the disclosure provides a method of treating or delaying progression of a cancer in a subject, the method comprising administering to the subject an effective amount of an RLR agonist provided by the disclosure, or a pharmaceutical composition provided by the disclosure.

In some aspects, the disclosure provides a method of reducing or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist provided by the disclosure, or a pharmaceutical composition provided by the disclosure.

In some aspects, the disclosure provides a method for stimulating an immune response, treating or delaying progression of a cancer, or inhibiting tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an RLR agonist provided by the disclosure, or a pharmaceutical composition provided by the disclosure, wherein the agonist, or the pharmaceutical composition increases RLR-mediated production of one or more cytokines in a cell, increases RLR-mediated expression of one or more interferon-stimulated genes in a cell, and or increases RLR-dependent intracellular signaling in a cell, thereby stimulating the immune response, treating or delaying progression of the cancer, or inhibiting growth of the tumor.

Combinations of RLR Agonists with Additional Therapeutic Agents

In some embodiments, an RLR agonist described herein can be administered to a subject as a monotherapy. Alternatively, the RLR agonist can be administered to a subject as a combination therapy with another treatment, e.g., another treatment for a cancer. For example, the combination therapy can include administering to the subject (e.g., a human patient) one or more additional agents that provide a therapeutic benefit to a subject who has, or is at risk of developing, cancer.

In some embodiments of the methods provided by the disclosure, the RLR agonist or pharmaceutical composition is administered in combination with one or more additional therapeutic agents, wherein the one or more additional therapeutic agents is selected from the group consisting of: a chemotherapy, a targeted anti-cancer therapy, an oncolytic drug, a cell death-inducing agent, an opsonizing agent (e.g., an opsonizing antibody) a cytotoxic agent, an immune-based therapy, a cytokine, an activator or agonist of a costimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine, a cellular immunotherapy, or a combination thereof.

In some embodiments, the RLR agonist or pharmaceutical composition is administered preceding or subsequent to administration of the one or more additional therapeutic agents or wherein the one or more additional therapeutic agents is administered concurrently with, preceding or subsequent to the administration of the agonist or pharmaceutical composition.

In some embodiments, the one or more additional therapeutic agents is a PD-1/PD-L1 antagonist, a TIM-3 antagonist, a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, a KIR antagonist, a LAG-3 antagonist, a toll-like receptor 3 (TLR3) agonist, a toll-like receptor 7 (TLR7) agonist, a toll-like receptor 9 (TLR9) agonist.

Combination with Chemotherapeutic Agents

Chemotherapeutic agents suitable for combination and/or co-administration with compositions of the present invention include, for example: taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxyanthrancindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Further agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioTEPA, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlordiamine platinum (II) (DDP), procarbazine, altretamine, cisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, or triplatin tetranitrate), anthracycline (e.g. daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. dactinomcin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g. vincristine and vinblastine) and temozolomide.

Combination with PD-1/PD-L1 Antagonists

In some embodiments, a RLR agonist, or pharmaceutical compositions thereof, provided by the disclosure is combined (e.g., administered in combination) with one or more PD-1/PD-L1 antagonist that specifically binds to human PD-1 or PD-L1 and inhibits PD-1/PD-L1 biological activity and/or downstream pathway(s) and/or cellular processed mediated by human PD-1/PD-L1 signaling or other human PD-1/PD-L1-mediated functions.

Accordingly, provided herein are PD-1/PD-L1 antagonists that directly or allosterically block, antagonize, suppress, inhibit or reduce PD-1/PD-L1 biological activity, including downstream pathways and/or cellular processes mediated by PD-1/PD-L1 signaling, such as receptor binding and/or elicitation of a cellular response to PD-1/PD-L1. Also provided herein are PD-1/PD-L1 antagonists that reduce the quantity or amount of human PD-1 or PD-L1 produced by a cell or subject.

In some embodiments, the disclosure provides a PD-1/PD-L1 antagonist that binds human PD-1 and prevents, inhibits or reduces PD-L1 binding to PD-1. In some aspects, the PD-1/PD-L1 antagonist binds to the mRNA encoding PD-1 or PD-L1 and prevents translation. In some embodiments, the PD-1/PD-L1 antagonist binds to the mRNA encoding PD-1 or PD-L1 and causes degradation and/or turnover.

In some embodiments, the PD-1/PD-L1 antagonist inhibits PD-1 signaling or function. In some embodiments, the PD-1/PD-L1 antagonist blocks binding of PD-1 to PD-L1, PD-L2, or to both PD-L1 and PD-L2. In some embodiments, the PD-1/PD-L1 antagonist blocks binding of PD-1 to PD-L1. In some embodiments, the PD-1/PD-L1 antagonist blocks binding of PD-1 to PD-L2. In some embodiments, the PD-1/PD-L1 antagonist blocks the binding of PD-1 to PD-L1 and PD-L2. In some embodiments, the PD-1/PD-L1 antagonist specifically binds PD-1. In some embodiments, the PD-1/PD-L1 antagonist specifically binds PD-L1. In some embodiments, the PD-1/PD-L1 antagonist specifically binds PD-L2.

In some embodiments, the PD-1/PD-L1 antagonist inhibits the binding of PD-1 to its cognate ligand. In some embodiments, the PD-1/PD-L1 antagonist inhibits the binding of PD-1 to PD-L1, PD-1 to PD-L2, or PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1/PD-L1 antagonist does not inhibit the binding of PD-1 to its cognate ligand.

In some embodiments, the PD-1/PD-L1 antagonist is an isolated monoclonal antibody (mAb), or antigen binding fragment thereof, which specifically binds to PD-1 or PD-L1. In some embodiments, the PD-1/PD-L1 antagonist is an antibody or antigen binding fragment thereof that specifically binds to human PD-1. In some embodiments, the PD-1/PD-L1 antagonist is an antibody or antigen binding fragment thereof that specifically binds to human PD-L1. In some embodiments, the PD-1/PD-L1 antagonist is an antibody or antigen binding fragment that binds to human PD-L1 and inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-1/PD-L1 antagonist is an antibody or antigen binding fragment that binds to human PD-1 and inhibits the binding of PD-L1 to PD-1.

Several immune checkpoint antagonists that inhibit or disrupt the interaction between PD-1 and either one or both of its ligands PD-L1 and PD-L2 are in clinical development or are currently available to clinicians for treating cancer.

Examples of anti-human PD-1 monoclonal antibodies, or antigen binding fragments thereof, that may comprise the PD-1/PD-L1 antagonist in any of the compositions, methods, and uses provided by the disclosure include, but are not limited to: KEYTRUDA® (pembrolizumab, MK-3475, h409A11; see U.S. Pat. Nos. 8,952,136, 8,354,509, 8,900,587, and EP2170959, all of which are included herein by reference in their entirety; Merck), OPDIVO® (nivolumab, BMS-936558, MDX-1106, ONO-4538; see U.S. Pat. Nos. 7,5950,48, 8,728,474, 9,073,994, 9,067,999, EP1537878, U.S. Pat. Nos. 8,008,449, 8,779,105, and EP2161336, all of which are included herein by reference in their entirety; Bristol Myers Squibb), MEDI0680 (AMP-514), BGB-A317 and BGB-108 (BeiGene), 244C8 and 388D4 (see WO2016106159, which is incorporated herein by reference in its entirety; Enumeral Biomedical), PDR001 (Novartis), and REGN2810 (Regeneron). Accordingly, in some embodiments the PD-1/PD-L1 antagonist is pembrolizumab. In some embodiments, the PD-1/PD-L1 antagonist is nivolumab.

Examples of anti-human PD-L1 monoclonal antibodies, or antigen binding fragments thereof, that may comprise the PD-1/PD-L1 antagonist in any of the compositions, methods, and uses provided by the disclosure include, but are not limited to: BAVENCIO® (avelumab, MSB0010718C, see WO2013/79174, which is incorporated herein by reference in its entirety; Merck/Pfizer), IMFINZI® (durvalumab, MEDI4736), TECENTRIQ® (atezolizumab, MPDL3280A, RG7446; see WO2010/077634, which is incorporated herein by reference in its entirety; Roche), MDX-1105 (BMS-936559, 12A4; see U.S. Pat. No. 7,943,743 and WO2013/173223, both of which are incorporated herein by reference in their entirety; Medarex/BMS), and FAZ053 (Novartis). Accordingly, in some embodiments the PD-1/PD-L1 antagonist is avelumab. In some embodiments, the PD-1/PD-L1 antagonist is durvalumab. In some embodiments, the PD-1/PD-L1 antagonist is atezolizumab.

In some embodiments, the PD-1/PD-L1 antagonist is an immunoadhesin that specifically bind to human PD-1 or human PD-L1, e.g., a fusion protein containing the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region such as an Fc region of an immunoglobulin molecule. Examples of immunoadhesion molecules that specifically bind to PD-1 are described in WO2010/027827 and WO2011/066342, both of which are incorporated herein by reference in their entirety. In some embodiments, the PD-1/PD-L1 antagonist is AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein that specifically binds to human PD-1.

It will be understood by one of ordinary skill that any PD-1/PD-L1 antagonist which binds to PD-1 or PD-L1 and disrupts the PD-1/PD-L1 signaling pathway, is suitable for compositions, methods, and uses disclosed herein.

In some embodiments, the PD-1/PD-L1 antagonist is a small molecule, a nucleic acid, a peptide, a peptide mimetic, a protein, a carbohydrate, a carbohydrate derivative, or a glycopolymer. Exemplary small molecule PD-1 inhibitors are described in Zhan et al., (2016) Drug Discov Today 21(6):1027-1036.

In some embodiments of the methods provided by the disclosure, the RLR agonist is combined with a PD-1/PD-L1 antagonist, wherein the PD-1/PD-L1 antagonist is selected from the group consisting of: PDR001, KEYTRUDA® (pembrolizumab), OPDIVO® (nivolumab), pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some embodiments, the PD-1/PD-L1 antagonist is selected from the group consisting of: FAZ053, TENCENTRIQ® (atezolizumab), BAVENCIO® (avelumab), IMFINZI® (durvalumab), and BMS-936559.

Combinations with TIM-3 Antagonist

In some embodiments, an RLR agonist, or pharmaceutical compositions thereof, provided by the disclosure is combined (e.g., administered in combination) with a TIM-3 antagonist. The TIM-3 antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the TIM-3 antagonist is chosen from MGB453 (Novartis), TSR-022 (Tesaro), or LY3321367 (Eli Lilly).

Combinations with LAG-3 Antagonist

In some embodiments, an RLR agonist, or pharmaceutical compositions thereof, provided by the disclosure is combined (e.g., administered in combination) with a LAG-3 antagonist. The LAG-3 antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. In some embodiments, the LAG-3 inhibitor is chosen from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck & Co), or REGN3767 (Regeneron).

Combinations with Toll-Like Receptor (TLR) Agonists

In some embodiments, an RLR agonist, or pharmaceutical composition thereof, provided by the disclosure is combined (e.g. administered in combination) with a TLR agonist.

Toll-like receptors (TLRs) are a family of germline-encoded transmembrane proteins that facilitate pathogen recognition and activation of the innate immune system. (Hoffmann et al., (1999) Science 284:1313-1318; Rock et al., (1998) Proc Natl Acad Sci USA 95:588-593). TLRs are pattern recognition receptors (PRRs), and are expressed by cells of the innate immune system. Examples of known ligands for TLRs include gram positive bacteria (TLR-2), bacterial endotoxin (TLR-4), flagellin protein (TLR-5), bacterial DNA (TLR-9), double-stranded RNA and poly I:C (TLR-3), and yeast (TLR-2). In vivo activation of TLRs initiates an innate immune response involving specific cytokines, chemokines and growth factors. While all TLRs can activate certain intracellular signaling molecules such as nuclear factor kappa beta (NF-κB) and mitogen activated protein kinases (MAP kinases), the specific set of cytokines and chemokines released appears to be unique for each TLR. TLR7, 8, and 9 comprise a subfamily of TLRs which are located in endosomal or lysosomal compartments of immune cells such as dendritic cells and monocytes. In contrast to TLR7 and 9 which are highly expressed on plasmacytoid dendritic cells (pDC), TLR8 is mainly expressed on myeloid DC (mDC) and monocytes. This subfamily mediates recognition of microbial nucleic acids, such as single stranded RNA.

Small, low-molecular weight (less than 400 Daltons) synthetic imidazoquinoline compounds which resemble the purine nucleotides adenosine and guanosine were the first TLR7 and TLR8 agonists to be identified. A number of these compounds have demonstrated anti-viral and anti-cancer properties. For example, the TLR7 agonist imiquimod (ALDARA™) was approved by the U.S. Food and Drug Administration as a topical agent for the treatment of skin lesions caused by certain strains of the human papillomavirus. Imiquimod may also be useful for the treatment of primary skin cancers and cutaneous tumors such as basal cell carcinomas, keratoacanthomas, actinic keratoses, and Bowen's disease. The TLR7/8 agonist resiquimod (R-848) is being evaluated as a topical agent for the treatment of human genital herpes.

TLR agonists according to the disclosure can be any TLR agonist. For example, a TLR agonist can encompass a natural or synthetic TLR ligand, a mutein or derivative of a TLR ligand, a peptide mimetic of a TLR ligand, a small molecule that mimics the biological function of a TLR ligand, or an antibody that stimulates a TLR receptor. A TLR ligand is any molecule that binds to a TLR.

In some embodiments, an RLR agonist, or pharmaceutical composition thereof, provided by the disclosure, is combined with a TLR agonist, wherein the TLR agonist is selected from the group consisting of: a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR10 agonist, and a TLR11 agonist.

In some embodiments, an RLR agonist provided by the disclosure is combined with a TLR3 agonist. A TLR3 agonist is an agonist that causes a signaling response through TLR3. Exemplary TLR3 agonists include, but are not limited to, polyinosinic:polycytidylic acid (poly I:C), HILTONOL® (poly ICLC), polyadenylic-polyuridylic acid (poly A:U), RIBOXXIM® (RGIC®100), RIBOXXON® (RGIC®50 bioconjugate), and RIBOXXOL® (RGIC®50).

In some embodiments, an RLR agonist provided by the disclosure is combined with polyinosinic:polycytidylic acid (poly I:C). In some embodiments, the RLR agonist is combined with HILTONOL® (poly ICLC). In some embodiments, the RLR agonist is combined with polyadenylic-polyuridylic acid (poly A:U). In some embodiments, the RLR agonist is combined with RIBOXXIM® (RGIC®100). In some embodiments, the RLR agonist is combined with RIBOXXON® (RGIC®50 bioconjugate). In some embodiments, the RLR agonist is combined with RIBOXXOL® (RGIC®50).

In some embodiments, an RLR agonist provided by the disclosure is combined with a TLR7 agonist. A TLR7 agonist is an agonist that causes a signaling response through TLR7. Non-limiting examples of TLR7 agonists include single stranded RNA (ssRNA), loxoribine (a guanosine analogue derivatized at positions N7 and C8), imidazoquinoline compounds (e.g., imiquimod and resiquimod), or derivatives thereof. Further exemplary TLR7 agonists include, but are not limited to, GS-9620 (Vesatolimod), imiquimod (ALDARA™), and resiquimod (R-848).

In some embodiments, an RLR agonist provided by the disclosure is combined with GS-9620 (Vesatolimod). In some embodiments, the RLR agonist is combined with imiquimod (ALDARA™). In some embodiments, the RLR agonist is combined with resiquimod (R-848).

In some embodiments, an RLR agonists provided by the disclosure is combined with a TLR9 agonist. A TLR9 agonist is an agonist that causes a signaling response through TLR9. Exemplary TLR9 agonists include, but are not limited to, CpG oligodeoxynucleotides (GpG ODNs). In some embodiments, the CpG ODN is a Class A CpG ODN (CpG-A ODN), a Class B CpG ODN (CpG-B ODN), or a Class C CpG ODN (CpG-B ODN).

In some embodiments, an RLR agonist provided by the disclosure is combined with a CpG oligodeoxynucleotide (CpG ODN). In some embodiments, the CpG ODN is a Class A CpG ODN (CpG-A ODN). In some embodiments, the CpG ODN is a Class B CpG ODN (CpG-B ODN). In some embodiments, the CpG ODN is a Class C CpG ODN (CpG-C ODN).

Other Combinations

In some embodiments, an RLR agonist, or pharmaceutical compositions thereof, provided by the disclosure is combined (e.g., administered in combination) with a VISTA antagonist, an adenosine A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a CTLA-4 antagonist, an IDO antagonist, or a KIR antagonist

In some embodiments, an RLR agonist, or pharmaceutical compositions thereof, provided by the disclosure is combined (e.g., administered in combination) with an agonist comprising an polypeptide (e.g, antibody, or antigen binding portion thereof) that specifically binds to CD137 (4-1BB).

In some embodiments, an RLR agonist, or pharmaceutical compositions thereof, provided by the disclosure is combined (e.g., administered in combination) with an agonist comprising an polypeptide (e.g., antibody, or antigen binding portion thereof) that specifically binds to CD134 (OX40).

An RLR agonist described herein can replace or augment a previously or currently administered therapy. For example, upon treating with an RLR agonist, administration of the one or more additional active agents can cease or diminish, e.g., be administered at lower levels or dosages. In some embodiments, administration of the previous therapy can be maintained. In some embodiments, a previous therapy will be maintained until the level of the RLR agonist reaches a level sufficient to provide a therapeutic effect. The two therapies can be administered in combination.

Monitoring a subject (e.g., a human patient) for an improvement in a cancer, as defined herein, means evaluating the subject for a change in a disease parameter, e.g., a reduction in tumor growth. In some embodiments, the evaluation is performed at least one (1) hour, e.g., at least 2, 4, 6, 8, 12, 24, or 48 hours, or at least 1 day, 2 days, 4 days, 10 days, 13 days, 20 days or more, or at least 1 week, 2 weeks, 4 weeks, 10 weeks, 13 weeks, 20 weeks or more, after an administration. The subject can be evaluated in one or more of the following periods: prior to beginning of treatment; during the treatment; or after one or more elements of the treatment have been administered. Evaluation can include evaluating the need for further treatment, e.g., evaluating whether a dosage, frequency of administration, or duration of treatment should be altered. It can also include evaluating the need to add or drop a selected therapeutic modality, e.g., adding or dropping any of the treatments for a cancer described herein.

In some embodiments, an RLR agonist described herein is administered to modulate a T-cell response in a patient, for example, by increasing T-cell activation and/or proliferation. Enhancement of T cell proliferation, IFN production and secretion, and/or the cytolytic activity of T cells may be beneficial to patients in need thereof to treat a disease or condition. Accordingly, in some embodiments, an RLR agonist of the present disclosure is administered to a patent in need thereof to induce or increase T-cell activation, enhance T cell proliferation, induce the production and/or secretion of IFN, and/or induce a cytolytic T cell response.

In some embodiments, an RLR agonist described herein, can be employed in methods of detection and/or quantification of human RLRs in a biological sample. Accordingly, an RLR agonist, as described herein, is used to diagnose, prognose, and/or determine progression of disease (e.g., cancer) in a patient.

While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

EXAMPLES

The disclosure will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1 Transfection of HuPBMCs with RLR Agonists Induces Cytokine Production In Vitro

To determine the effect of RLR agonists comprising various modification on cytokine induction, the ability of RLR agonists to induce cytokine production was assessed in vitro. Human peripheral blood mononuclear cells (huPBMCs) were prepared from two healthy donors and seeded at a density of 2×10⁵ cells/well in a standard 96-well tissue culture plate in 100 μL of RPMI 1640 cell culture medium supplemented with fetal calf serum (FCS), L-glutamine, and Pen/Strep. Independent transfections of huPBMCs with RLR agonists, as indicated in FIG. 1, was carried out using Lipofectamine2000 as the transfection reagent (except for G10 and ODN2216, where direct incubation was applied, data not shown). Cells were incubated for 24 h at 37C in a humidified incubator followed by harvesting of cell culture supernatant. Supernatants were immediately frozen and stored at −20C. Samples were thawed once for analysis of cytokines IFN-α2a (FIG. 1), as well as IL-1β, IP-10, IL-6, IL-12p70, MCP-1 and MIP-1β (data not shown) using a U-Plex MSD platform according to the manufacturer's instructions. FIG. 1 shows the dose-dependent induction of IFN-α secretion from human PBMC treated with novel candidate RLR agonists comprising various modifications and/or sequence motifs. RLR agonists we added at either 10 nM, 2 nM, or 0.4 nM. The amount of IFN-α2a released by the cells in response to RLR agonist transfection is given in pg/mL.

Tables 3 and 4 show the sequences of each RLR agonist. Table 3 also shows the sequence and number corresponding to each compound tested in FIG. 1. For examples, compound X25224 in FIG. 1 corresponds to “RIG7” which comprises a first oligonucleotide comprising SEQ ID NO: 42 linked via a linker “UUCG” to a second oligonucleotide comprising SEQ ID NO: 73 and has a 5′ diphosphate moiety. The sequence of RIG7 is also set forth as SEQ ID NO: 6 in Table 4.

TABLE 3 OLIGONUCLEOTIDE COMBINATION TABLE First Second Oligonu- Oli- 5′ cleotide gonucleotide Phosphate RNA (FO) (SO) Linker Moiety RIG 2a (X32671) 37 68 UUCG — RIG 2b (X25217) 37 68 UUCG pp RIG 3a (X32666) 38 69 UUCG — RIG 3b (X25218) 38 69 UUCG pp RIG 4 (X25219) 39 70 UUCG pp RIG 5 (X25221) 40 71 UUCG pp RIG 6 (X25222) 41 72 UUCG pp RIG 7 (X25224) 42 73 UUCG pp RIG 8 (X25225) 43 74 UUCG pp RIG 9 (X25226) 44 75 UUCG pp RIG 10 (X25227) 45 76 UUCG pp RIG 11 (X25228) 46 77 UUCG pp RIG 12 (X25229) 47 78 UUCG pp RIG 13a (X32667) 48 79 UUCG — RIG 13b (X25230) 48 79 UUCG pp RIG 13c (X24921) 48 79 UUCG ppp RIG 14 (X25231) 49 80 UUCG pp RIG 15a (X32665) 50 81 UUCG — RIG 15b (X25232) 50 81 UUCG pp RIG 15c (X24923) 50 81 UUCG ppp RIG 16 (X25233) 51 82 UUCG pp RIG 18 (X25234) 52 83 UUCG pp RIG 20a (X32750) 53 84 UUCG — RIG 20b (X25235) 53 84 UUCG pp RIG 21 (X25237) 54 85 UUCG pp RIG 22a (X32672) 55 86 UUCG — RIG 22b (X25239) 55 86 UUCG pp RIG 24a (X25241) 56 87 UUCG pp RIG 24b (X25240) 56 87 UUCG ppp RIG 25 (X25242) 57 88 UUCG pp RIG 26 (X25243) 58 89 UUCG pp RIG 27a (X32669) 59 89 UUCG — RIG 27b (X25244) 59 89 UUCG pp RIG 27c (X24935) 59 89 UUCG ppp RIG 28a (X25245) 60 90 UUCG pp RIG 28b (X24936) 60 90 UUCG ppp RIG 35a (X32670) 61 91 UUCG — RIG 35b (X25247) 61 91 UUCG pp RIG 36 (X24945) 62 92 UUCG pp RIG 37a (X25249) 63 91 UUUGAU pp RIG 37b (X25248) 63 91 UUUGAU ppp RIG 38a (X32668) 63 91 UGUUU — RIG 38b (X25251) 63 91 UGUUU pp RIG 39 (X25253) 63 91 GAUC pp RIG 40 (X25255) 64 93 GAUC pp RIG 41 (X25257) 65 94 GAUC pp RIG 42 (X25259) 64 93 UUCG pp RIG 43a (X32673) 63 91 (C9) — RIG 43b (X25261) 63 91 (C9) pp RIG 44 (X25263) 63 91 (HEG) pp RIG 47 (X25265) 66 95 UUCG pp RIG 48 (X25267) 67 96 UUCG pp RIG 49a (X25269) 63 97 UUCG pp RIG 49b (X25268) 63 97 UUCG ppp RIG 50a (14L) 63 91 UUCG — (X32664) RIG 50b (14L) 63 91 UUCG pp (X24943) RIG 50c (14L) 63 91 UUCG ppp (X24907) (—) indicates no 5′ phosphate; (pp) indicates 5′ diphosphate; (ppp) indicates 5′ triphosphate

TABLE 4 SEQUENCE LISTING SEQ ID NO Description Sequence   1 RIG 2 GGATCGATCGATCGUUCGCGATCGATCGATCC Nucleic  acid sequence   2 RIG 3 GGAUCGAUCGAUAUUUCGAUAUCGAUCGAUCC Nucleic  acid sequence   3 RIG 4 GCGCGCGCGCGCGCUUCGGCGCGCGCGCGCGC Nucleic  acid sequence   4 RIG 5 GGCGGCGCGCCGCCUUCGGGCGGCGCGCCGCC Nucleic  acid sequence   5 RIG 6 GGCGGCGGCGGCGGUUCGCCGCCGCCGCCGCC Nucleic  acid sequence   6 RIG 7 GGCGGCCGCCCGCGUUCGCGCGGGCGGCCGCC Nucleic  acid sequence   7 RIG 8 CGACGUCGACGUCGUUCGCGACGUCGACGUCG Nucleic  acid sequence   8 RIG 9 GCACGUCGACGUGCUUCGGCACGUCGACGUGC Nucleic  acid sequence   9 RIG 10 GGACGUCGACGUCCUUCGGGACGUCGACGUCC Nucleic  acid sequence  10 RIG 11 GGUCGCGACCAUAUUUCGAUAUGGUCGCGACC Nucleic  acid sequence  11 RIG 12 GGAUACGUCGACGUUUCGACGUCGACGUAUCC Nucleic  acid sequence  12 RIG 13 GAGAGAGAGAGAGAUUCGUCUCUCUCUCUCUC Nucleic  acid sequence  13 RIG 14 GAGUCUAGACUCCGUUCGCGGAGUCUAGACUC Nucleic  acid sequence  14 RIG 15 CGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCG Nucleic  acid sequence (RIG 45)  15 RIG 16 CCAUCGAUCGAUCGUUCGCGAUCGAUCGAUGG Nucleic  acid sequence  16 RIG 18 GAAUCGAUCGAUCGUUCGCGAUCGAUCGAUUC Nucleic  acid sequence  17 RIG 20 GGGAUCGAUCGUUCGCGAUCGAUCCC Nucleic  acid sequence  18 RIG 21 CCCCCGAUCGAUCGUUCGCGAUCGAUCGGGGG Nucleic  acid sequence  19 RIG 22 GTGTGTGTGTGTGTUUCGACACACACACACAC Nucleic  acid sequence  20 RIG 24 GTGTGTGGAUCGAUUUCGAUCGAUCCACACAC Nucleic  acid sequence  21 RIG 25 GGAICGAICGAICGUUCGCGAICGAICGAICC Nucleic  acid sequence  22 RIG 26 IIAUCIAUCIAUCIUUCGCIAUCIAUCIAUCC Nucleic  acid sequence  23 RIG 27 GGAUCIAUCIAUCIUUCGCIAUCIAUCIAUCC Nucleic  acid sequence  24 RIG 28 GGIUCGIUCGIUCGUUCGCGIUCGIUCGIUCC Nucleic  acid sequence  25 RIG 35 IGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCC Nucleic  acid sequence  26 RIG 36 AUCGAUCGAUCGUUCGCGAUCGAUCGAU Nucleic  acid sequence  27 RIG 37 GGAUCGAUCGAUCGUUUGAUCGAUCGAUCGAU Nucleic  CC acid sequence  28 RIG 38 GGAUCGAUCGAUCGUGUUUCGAUCGAUCGAUC Nucleic  C acid sequence  29 RIG 39 GGAUCGAUCGAUCGGAUCCGAUCGAUCGAUCC Nucleic  acid sequence  30 RIG 40 GGCAUGCGACCUCUGUUUGAUCAAACAGAGGU Nucleic  CGCAUGCC acid sequence  31 RIG 41 GGCAUGCGACCUCUGAUCAGAGGUCGCAUGCC Nucleic  acid sequence  32 RIG 42 GGCAUGCGACCUCUGUUUUUCGAAACAGAGGU Nucleic  CGCAUGCC acid sequence  33 RIG 47 TGCUCGAUCGAUCGUUCGCGAUCGAUCGAGCA Nucleic  acid sequence  34 RIG 48 TCGUCGAUCGAUCGUUCGCGAUCGAUCGACGA Nucleic  acid sequence  35 RIG 49 GGAUCGAUCGAUCGUUCGTGAUCGAUCGAUGG Nucleic  acid sequence  36 RIG 50 GGAUCGAUCGAUCGUUCGCGAUCGAUCGAUCC Nucleic  acid sequence (14L)  37 FO1 GGATCGATCGATCG Nucleic  acid sequence  38 FO2 GGAUCGAUCGAUAU Nucleic  acid sequence  39 FO3 GCGCGCGCGCGCGC Nucleic  acid sequence  40 FO4 GGCGGCGCGCCGCC Nucleic  acid sequence  41 FO5 GGCGGCGGCGGCGG Nucleic  acid sequence  42 FO6 GGCGGCCGCCCGCG Nucleic  acid sequence  43 FO7 CGACGUCGACGUCG Nucleic  acid sequence  44 FO8 GCACGUCGACGUGC Nucleic  acid sequence  45 FO9 GGACGUCGACGUCC Nucleic  acid sequence  46 FO10 GGUCGCGACCAUAU Nucleic  acid sequence  47 FO11 GGAUACGUCGACGU Nucleic  acid sequence  48 FO12 GAGAGAGAGAGAGA Nucleic  acid sequence  49 FO13 GAGUCUAGACUCCG Nucleic  acid sequence  50 FO14 CGAUCGAUCGAUCG Nucleic  acid sequence  51 FO15 CCAUCGAUCGAUCG Nucleic  acid sequence  52 FO16 GAAUCGAUCGAUCG Nucleic  acid sequence  53 FO17 GGGAUCGAUCG Nucleic  acid sequence  54 FO18 CCCCCGAUCGAUCG Nucleic  acid sequence  55 FO19 GTGTGTGTGTGTGT Nucleic  acid sequence  56 FO20 GTGTGTGGAUCGAU Nucleic  acid sequence  57 FO21 GGAICGAICGAICG Nucleic  acid sequence  58 FO22 IIAUCIAUCIAUCI Nucleic  acid sequence  59 FO23 GGAUCIAUCIAUCI Nucleic  acid sequence  60 FO24 GGIUCGIUCGIUCG Nucleic  acid sequence  61 FO25 IGAUCGAUCGAUCG Nucleic  acid sequence  62 FO26 AUCGAUCGAUCG Nucleic  acid sequence  63 FO27 GGAUCGAUCGAUCG Nucleic  acid sequence  64 FO28 GGCAUGCGACCUCUGUUU Nucleic  acid sequence  65 FO29 GGCAUGCGACCUCU Nucleic  acid sequence  66 FO30 TGCUCGAUCGAUCG Nucleic  acid sequence  67 FO31 TCGUCGAUCGAUCG Nucleic  acid sequence  68 SO1 CGATCGATCGATCC Nucleic  acid sequence  69 SO2 AUAUCGAUCGAUCC Nucleic  acid sequence  70 SO3 GCGCGCGCGCGCGC Nucleic  acid sequence  71 SO4 GGCGGCGCGCCGCC Nucleic  acid sequence  72 SO5 CCGCCGCCGCCGCC Nucleic  acid sequence  73 SO6 CGCGGGCGGCCGCC Nucleic  acid sequence  74 SO7 CGACGUCGACGUCG Nucleic  acid sequence  75 SO8 GCACGUCGACGUGC Nucleic  acid sequence  76 SO9 GGACGUCGACGUCC Nucleic  acid sequence  77 SO10 AUAUGGUCGCGACC Nucleic  acid sequence  78 SO11 ACGUCGACGUAUCC Nucleic  acid sequence  79 SO12 UCUCUCUCUCUCUC Nucleic  acid sequence  80 SO13 CGGAGUCUAGACUC Nucleic  acid sequence  81 SO14 CGAUCGAUCGAUCG Nucleic  acid sequence  82 SO15 CGAUCGAUCGAUGG Nucleic  acid sequence  83 SO16 CGAUCGAUCGAUUC Nucleic  acid sequence  84 SO17 CGAUCGAUCCC Nucleic  acid sequence  85 SO18 CGAUCGAUCGGGGG Nucleic  acid sequence  86 SO19 ACACACACACACAC Nucleic  acid sequence  87 SO20 AUCGAUCCACACAC Nucleic  acid sequence  88 SO21 CGAICGAICGAICC Nucleic  acid sequence  89 SO22 CIAUCIAUCIAUCC Nucleic  acid sequence  90 SO23 CGIUCGIUCGIUCC Nucleic  acid sequence  91 SO24 CGAUCGAUCGAUCC Nucleic  acid sequence  92 SO25 CGAUCGAUCGAU Nucleic  acid sequence  93 SO26 AAACAGAGGUCGCAUGCC Nucleic  acid sequence  94 SO27 AGAGGUCGCAUGCC Nucleic  acid sequence  95 SO28 CGAUCGAUCGAGCA Nucleic  acid sequence  96 SO29 CGAUCGAUCGACGA Nucleic  acid sequence  97 SO30 TGAUCGAUCGAUGG Nucleic  acid sequence  98 Human MTTEQRRSLQAFQDYIRKTLDPTYILSYMAPW RIG-I FREEEVQYIQAEKNNKGPMEAATLFLKFLLEL Amino  QEEGWFRGFLDALDHAGYSGLYEAIESWDFKK acid IEKLEEYRLLLKRLQPEFKTRIIPTDIISDLS sequence ECLINQECEEILQICSTKGMMAGAEKLVECLL RSDKENWPKTLKLALEKERNKFSELWIVEKGI KDVETEDLEDKMETSDIQIFYQEDPECQNLSE NSCPPSEVSDTNLYSPFKPRNYQLELALPAMK GKNTIICAPTGCGKTFVSLLICEHHLKKFPQG QKGKVVFFANQIPVYEQQKSVFSKYFERHGYR VTGISGATAENVPVEQIVENNDIIILTPQILV NNLKKGTIPSLSIFTLMIFDECHNTSKQHPYN MIMFNYLDQKLGGSSGPLPQVIGLTASVGVGD AKNTDEALDYICKLCASLDASVIATVKHNLEE LEQVVYKPQKFFRKVESRISDKFKYIIAQLMR DTESLAKRICKDLENLSQIQNREFGTQKYEQW IVTVQKACMVFQMPDKDEESRICKALFLYTSH LRKYNDALIISEHARMKDALDYLKDFFSNVRA AGFDEIEQDLTQRFEEKLQELESVSRDPSNEN PKLEDLCFILQEEYHLNPETITILFVKTRALV DALKNWIEGNPKLSFLKPGILTGRGKTNQNTG MTLPAQKCILDAFKASGDHNILIATSVADEGI DIAQCNLVILYEYVGNVIKMIQTRGRGRARGS KCFLLTSNAGVIEKEQINMYKEKKMNDSILRL QTWDEAVFREKILHIQTHEKFIRDSQEKPKPV PDKENKKLLCRKCKALACYTADVRVIEECHYT VLGDAFKECFVSRPHPKPKQFSSFEKRAKIFC ARQNCSHDWGIHVKYKTFEIPVIKIESFVVED IATGVQTLYSKWKDFHFEKIPFDPAEMSK  99 Human MSNGYSTDENFRYLISCFRARVKMYIQVEPVL MDA5 DYLTFLPAEVKEQIQRTVATSGNMQAVELLLS Amino  TLEKGVWHLGWTREFVEALRRTGSPLAARYMN acid PELTDLPSPSFENAHDEYLQLLNLLQPTLVDK sequence LLVRDVLDKCMEEELLTIEDRNRIAAAENNGN ESGVRELLKRIVQKENWFSAFLNVLRQTGNNE LVQELTGSDCSESNAEIENLSQVDGPQVEEQL LSTTVQPNLEKEVWGMENNSSESSFADSSVVS ESDTSLAEGSVSCLDESLGHNSNMGSDSGTMG SDSDEENVAARASPEPELQLRPYQMEVAQPAL EGKNIIICLPTGSGKTRVAVYIAKDHLDKKKK ASEPGKVIVLVNKVLLVEQLFRKEFQPFLKKW YRVIGLSGDTQLKISFPEVVKSCDIIISTAQI LENSLLNLENGEDAGVQLSDFSLIIIDECHHT NKEAVYNNIMRHYLMQKLKNNRLKKENKPVIP LPQILGLTASPGVGGATKQAKAEEHILKLCAN LDAFTIKTVKENLDQLKNQIQEPCKKFAIADA TREDPFKEKLLEIMTRIQTYCQMSPMSDFGTQ PYEQWAIQMEKKAAKEGNRKERVCAEHLRKYN EALQINDTIRMIDAYTHLETFYNEEKDKKFAV IEDDSDEGGDDEYCDGDEDEDDLKKPLKLDET DRFLMTLFFENNKMLKRLAENPEYENEKLTKL RNTIMEQYTRTEESARGIIFTKTRQSAYALSQ WITENEKFAEVGVKAHHLIGAGHSSEFKPMTQ NEQKEVISKFRTGKINLLIATTVAEEGLDIKE CNIVIRYGLVTNEIAMVQARGRARADESTYVL VAHSGSGVIEHETVNDFREKMMYKAIHCVQNM KPEEYAHKILELQMQSIMEKKMKTKRNIAKHY KNNPSLITFLCKNCSVLACSGEDIHVIEKMHH VNMTPEFKELYIVRENKALQKKCADYQINGEI ICKCGQAWGTMMVHKGLDLPCLKIRNFVVVFK NNSTKKQYKKWVELPITFPNLDYSECCLFSDE D 100 Human MELRSYQWEVIMPALEGKNIIIWLPTGAGKTR LGP2 AAAYVAKRHLETVDGAKVVVLVNRVHLVTQHG Amino  EEFRRMLDGRWTVTTLSGDMGPRAGFGHLARC acid HDLLICTAELLQMALTSPEEEEHVELTVFSLI sequence VVDECHHTHKDTVYNVIMSQYLELKLQRAQPL PQVLGLTASPGTGGASKLDGAINHVLQLCANL DTWCIMSPQNCCPQLQEHSQQPCKQYNLCHRR SQDPFGDLLKKLMDQIHDHLEMPELSRKFGTQ MYEQQVVKLSEAAALAGLQEQRVYALHLRRYN DALLIHDTVRAVDALAALQDFYHREHVTKTQI LCAERRLLALFDDRKNELAHLATHGPENPKLE MLEKILQRQFSSSNSPRGIIFTRTRQSAHSLL LWLQQQQGLQTVDIRAQLLIGAGNSSQSTHMT QRDQQEVIQKFQDGTLNLLVATSVAEEGLDIP HCNVVVRYGLLTNEISMVQARGRARADQSVYA FVATEGSRELKRELINEALETLMEQAVAAVQK MDQAEYQAKIRDLQQAALTKRAAQAAQRENQR QQFPVEHVQLLCINCMVAVGHGSDLRKVEGTH HVNVNPNFSNYYNVSRDPVVINKVFKDWKPGG VISCRNCGEVWGLQMIYKSVKLPVLKVRSMLL ETPQGRIQAKKWSRVPFSVPDFDFLQHCAENL SDLSLD 

1.-69. (canceled)
 70. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising the formula: 5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein (i) (N₁-N₂-X₁) comprises a first polynucleotide comprising linked nucleotides N₁, N₂ and X₁, (ii) (X₂-N₃-N₄) comprises a second polynucleotide comprising linked nucleotides X₂, N₃ and N₄; (iii) N₁, N_(2,) N₃ and N₄ each comprise a single nucleotide comprising a nucleoside selected from the group consisting of: adenosine, guanosine, cytidine, 5-methyluridine, uridine and inosine; (iv) N₁ base pairs with N₄; (v) N₂ base pairs with N₃; (vi) N₁ comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof; (vii) X₁ comprises a sequence motif [AUCN₅]_(x), wherein N₅ comprises guanosine or inosine, wherein x is an integer whose value indicates the number of sequence motifs, and wherein x=2 to 4; (viii) X₂ comprises a sequence motif [CN₆AU]_(y), wherein N6 comprises guanosine or inosine, wherein y is an integer whose value indicates the number of sequence motifs, and wherein y=2 to 4; (ix) L is a linker that operably links the first polynucleotide and the second polynucleotide, wherein at least one of N1, N2, N3, and N4 is inosine, and/or at least one of X1 and/or X2 comprises at least one inosine nucleoside, and wherein the inosine nucleoside base pairs with cytidine in the hairpin RNA.
 71. The agonist of claim 70, wherein N5 comprises inosine and N6 comprises inosine. 72.-74. (canceled)
 75. The agonist of claim 70, wherein x=3 and y=3. 76.-80. (canceled)
 81. The agonist of claim 70, wherein N1 comprises guanosine (G).
 82. The agonist of claim 70, wherein N2 comprises guanosine (G). 83.-84. (canceled)
 85. The agonist of claim 70, wherein N1 and N2 comprise guanosine (G) and N₃ and N₄ comprise cytidine (C). 86.-119. (canceled)
 120. The agonist of claim 70, wherein the linker is a nucleotide linker. 121-122. (canceled)
 123. The agonist of claim 70, wherein the nucleotide linker comprises a tetraloop comprising the sequence UUCG: 124-131. (canceled)
 132. The agonist of claim 70, wherein the agonist comprises a 5′ diphosphate moiety, or a derivative or analog thereof. 133.-139 (canceled)
 140. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, and wherein the agonist comprises the nucleotide sequence selected from the group consisting of SEQ ID NOs: 22, 23 and
 25. 141. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising at least one or more nucleotides comprising inosine which base pairs with cytidine, wherein the agonist comprises the formula 5′-(N₁-N₂-X₁)-L-(X₂-N₃-N₄)-3′, wherein (N₁-N₂-X₁) comprises a first polynucleotide and (X₂-N₃-N₄) comprises a second polynucleotide, and wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences selected from the group consisting of: (i) SEQ ID NO: 58 and 89, respectively; (ii) SEQ ID NO: 59 and 89, respectively; and (iii) SEQ ID NO: 61 and 91, respectively.
 142. (canceled)
 143. The agonist of claim 70, wherein the nucleotide sequence comprising the agonist is not complementary to a genomic DNA sequence or mRNA sequence, wherein the RLR agonist does not participate in RNA interference, and wherein the RLR agonist does not silence gene expression. 144.-146. (canceled)
 147. A method to increase RLR-mediated production of one or more cytokines in a cell, the method comprising contacting the cell with the agonist of claim 70, wherein the agonist increases RLR-mediated cytokine production in a cell. 148.-189. (canceled)
 190. A kit comprising the agonist of claim 70 and instructions for use in stimulating an immune response in a subject, or treating or delaying progression of a cancer, or inhibiting tumor growth in a subject, optionally with instructions for use in combination with one or more additional therapeutic agents. 191.-195. (canceled)
 196. The agonist of claim 70, wherein x=2 and y=2.
 197. The agonist of claim 140, wherein the agonist comprises the nucleotide sequence of SEQ ID NO:
 23. 198. The agonist of claim 147, wherein the first polynucleotide and the second polynucleotide comprise the nucleotide sequences SEQ ID NOs: 59 and 89, respectively.
 199. A synthetic RIG-I-like receptor (RLR) agonist that specifically binds to a RIG-I-like receptor (RLR), wherein the agonist comprises a blunt-ended, hairpin RNA comprising a first polynucleotide connected to a second polynucleotide by a linker, wherein the first polynucleotide is sufficiently complementary to the second polynucleotide to form a duplex, wherein the duplex comprises less than 19 base pairs, wherein the 5′ most nucleotide of the first oligonucleotide comprises a 5′ diphosphate or triphosphate moiety, or derivative or analog thereof; wherein the agonist comprises a [AUCG]n repeat motif, where n=2-4; and wherein the AUCG repeat motif is preceded by GG, CG, or IG.
 200. The agonist of claim 199, wherein n=2, and wherein the AUCG repeat motif is preceded by GG.
 201. The agonist of claim 199, wherein n=3, and wherein the AUCG repeat motif is preceded by CG.
 202. The agonist of claim 199, wherein n=3, and wherein the AUCG repeat motif is preceded by IG.
 203. The agonist of claim 199, wherein n=3, and wherein the at least one G comprising at least one AUCG motif is substituted by inosine. 